SSNIP-seq: A simple and rapid method for isolation of single-sperm nucleic acid for high-throughput sequencing.

We developed a simple and reliable method for the isolation of haploid nuclei from fresh and frozen testes. The described protocol uses readily available reagents in combination with flow cytometry to separate haploid and diploid nuclei. The protocol can be completed within 1 hour and the resulting individual haploid nuclei have intact morphology. The isolated nuclei are suitable for library preparation for high-throughput DNA and RNA sequencing using bulk or single nuclei. The protocol was optimised with mouse testes and we anticipate that it can be applied for the isolation of mature sperm from other mammals including humans.

Introduction

Background

Sperm can be used with genomic technologies to study diverse topics such as fertility, genome structure and forensic science [1-5]. A major challenge for the isolation of individual haploid cells from males for sequencing is that they stick to most pipette tips and tubes, and traditional methods for bulk sperm isolation use technically challenging density gradients. Therefore, the motivation for developing this technique is to have a simple and rapid nucleic acid preparation technique that is compatible with high-throughput sequencing technologies, including for bulk and single cell ATAC-seq, RNA-seq and similar approaches. Several studies have been published that focused on the sequencing of individual mouse or human sperm [6-11] highlighting the interest in the biology and the utility of a simple method that uses standard laboratory materials and equipment.

Development and application of the method

We have developed a simple and reliable protocol for the isolation of over 100,000 haploid nuclei derived from mouse testis, which we refer to as SSNIP-seq (single-sperm nuclei isolation protocol). The protocol is adapted from the ‘Frankenstein’ protocol for nuclei isolation [12] and makes modifications that have allowed us to reproducibly isolate over 100,000 haploid nuclei from a single mouse testis or epididymis in approximately 30 minutes (Fig 1, S1 Table). We use 100,000 as a minimum target number in this protocol as droplet-based single-nucleus library preparation methods can require a high concentration of nuclei in a small volume. This suspension is then used as the starting material for the protocol. It is important to include a diploid control sample. This control is used to define what is a diploid cell at G1 or G2 during cell sorting, and in turn what cells are haploid. This method is developed to isolate nuclei from both spermatids and spermatozoa. We use spleen as high numbers of cells can be put into suspension with very simple mechanical disruption. The resulting nuclei from all samples are stained with DAPI and sorted using flow cytometry to isolate the haploid nuclei from the testis sample, which can then be used as material for a variety of high-throughput sequencing techniques (Fig 1). We have successfully used these nuclei in bulk ATAC-seq, scATAC-seq and single nuclei CNV experiments for mouse haploid nuclei [13]. We propose that the protocol can be easily adapted for mature human sperm.

Fig 1

Schematic summary of the protocol.

The protocol workflow consists of three key parts: 1) tissue collection, 2) nuclei isolation and buffer exchange, and 3) cell sorting. This protocol results in the isolation of haploid sperm-derived nuclei which may then be utilised in a variety of downstream applications, including both plate- and droplet-based approaches.

Cell sorter (BD400 Class II FACSAria Fusion Cell Sorter with FACS Diva software, Becton Dickinson)

Immunofluorescence microscope that can visualise DAPI

Benchtop microcentrifuge with temperature control or located in a cold room. We use an Eppendorf Centrifuge 5418 in a 4 °C cold room.

PCR tube adapters for the microcentrifuge

Timer

Flowmi cell strainers, 40 μm (Bel-Art, cat no. H13680-0040)

DNA LoBind microcentrifuge tubes, 1.5 mL (Eppendorf, cat no. 0030 108 418)

Round-bottom polypropylene tubes with cap, 5 mL (Falcon, cat no. 352063)

Wide bore pipette tips, 1000 μL (Axygen, cat no. T-1005-WB-C) (optional)

Micropipettes DV1000, DV200, DV20, D10 (THL, cat no. PZ-7901)

Cell strainers, 70 μm (Corning, cat no. 431751)

Tweezers with pointed tips, 115mm (Weller Erem, cat no. 5SA)

Tissue-culture treated culture dishes, 35 x 10 mm (Corning, cat no. 430165)

Thin wall PCR tubes with flat cap, 200 μL (Axygen, cat no. PCR-02-L-C)

96-well plate with round bottom (Greiner, cat no. 650101)

Sterile syringe 0.2 μm filters, acrodisc (Pall, cat no. 2415600)

50 mL syringe (BD, cat no. 300144)

50 mL centrifuge tube (Sarstedt, cat no. 92.547.254)

2 mL plastic syringe (BD, cat. No. 302204)

Materials and methods

Biological material

One wild-type male mouse six weeks or older. This protocol was developed using C57BL/6J, FVB/N, and F1(C57BL/6J x FVB/N) mice.

10x Dulbecco’s Phosphate Buffered Saline (DPBS), without calcium chloride and magnesium chloride (Sigma Aldrich, cat no. D1408-500ML)

1x DPBS, without calcium chloride and magnesium chloride (Sigma Aldrich, cat no. D8537-500ML)

4, 6-Diamidino-2-phenylindole dihydrochloride (DAPI; Sigma Aldrich, cat no. D8417-1MG)

‘Bovostar’ Bovine Serum Albumin (BSA; Bovogen Biologicals, cat no. BSASAU-0.1)

Nuclei EZ Lysis Buffer from Nuclei EZ Prep Kit (Sigma Aldrich, cat no. NUC101-1KT)

DAPI stock–Prepare 1 mg / mL of DAPI in sterile water. Filtered using a 0.22 μm filter and stored at -20°C.

10% BSA–Prepare a 10% BSA (w/v) stock solution with sterile water. Filter using a 0.22 μm filter and store at 4°C for up to a month.

Nuclei Wash Resuspension Buffer (NWRB) with 1% or 0.1% BSA (w/v)–Prepare solution using 10x DBPS, 10% BSA stock and sterile water. Make fresh prior to use and keep on ice.

NWRB with 1% BSA and DAPI (10 μg / mL)–Prepare using 10x DBPS, 10% BSA stock, DAPI stock and sterile water. Make fresh prior to use, keep on ice and protect from light.

Procedure

All steps must be completed quickly, and samples kept at 4°C at all times. The use of Lo-Bind microcentrifuge tubes is critical to avoid excessive loss of sperm cells or nuclei.

1| Sacrifice mice using CO2 or cervical dislocation. Following local SOPs that have been approved by an animal ethics committee. All experimental procedures were approved in writing by the St. Vincent’s Hospital Melbourne Animal Ethics Committee.

2| Dissect testis and spleens from each mouse, removing as much fat and unwanted connective tissue as possible, and place each organ individually in a 1.5 mL microcentrifuge tube containing cold DPBS (Fig 1).

If nuclei isolation is performed on the day, place the testis on ice and begin the protocol within 30 minutes of dissection. Otherwise, the testes can be individually placed in an empty 1.5 mL microcentrifuge tube and snap frozen in liquid nitrogen then stored at -80°C. We have obtained >100,000 nuclei from frozen testis samples up to 1 month after freezing.

Spleen cell suspension

A spleen cell suspension is formed which will serve as a diploid control.

3| Place a 70 μm cell strainer into a 50 mL centrifuge tube and wet the strainer by pipetting 1 mL cold DPBS onto the strainer mesh.

4| Place the whole spleen into the strainer and homogenise it using the plunger seal of a 2 mL plastic syringe (S1 Fig). Rinse the strainer with an additional 1 mL of cold DPBS. Place the sample on ice until step 7.

The solution should look homogenous. If the suspension contains clumps of material, it can be filtered into a second 50 mL centrifuge tube using a clean 70 μm cell strainer.

Nuclei isolation

At this stage it is anticipated that you will have:

Either a fresh or frozen testis;

If using a frozen testis allow it to thaw on ice for 10 minutes; and

A spleen to serve as a diploid control

All steps are performed on ice

5| Place two 1.5 mL microcentrifuge tubes on ice and add 1 mL of chilled Nuclei EZ Lysis Buffer to each.

6| Transfer the fresh or thawed testis to one of the microcentrifuge tubes.

Release the seminiferous tubules from the testis by gently squeezing the testis with pointed tweezers until the testis burst. Gently tear and homogenise the testis and in turn the seminiferous tubules several times with the tweezers to further break them apart and allow the release of more cells into solution. The solution should be cloudy due to the high cell number.

7| Add 300 μL of the spleen cell suspension (from step 4) to the second microcentrifuge tube (Fig 1).

8| Incubate both the spleen and the testis samples on ice for 5 minutes to allow cell lysis and the release of nuclei. From here until step 18 (Cell sorting) both the testis and spleen sample are treated the same.

After 3 minutes of the 5-minute incubation gently invert the samples two or three times to allow better mixing of the cells with the Nuclei EZ Lysis Buffer.

For mutants with smaller testis or low sperm production it may be necessary to break the testis apart more extensively or break apart two testes in the same tube. To overcome volume constraints this may need to be performed in a 2 mL Lo-Bind microcentrifuge.

9| Just prior to the end of the 5-minute incubation period, remove any large pieces of non-degraded testis debris.

Failure to remove debris will result in unwanted clumping of debris and the target nuclei onto the bottom of the tube.

10| Centrifuge the samples at 500 x g for 5 minutes at 4°C. Remove the supernatant, leaving behind just enough to cover the pellet. Do not allow the pellet to dry.

11| Add 1 mL of Nuclei EZ Lysis Buffer. Repeat steps 8–10.

12| Very slowly add 1 mL of NWRB with 1% BSA and incubate the sample on ice for 5 minutes to allow buffer exchange. Take care to avoid resuspending the pellet.

13| After incubation gently resuspend the pellet by either inverting 5 times or pipetting up and down 5 times with a wide-bore tip.

14| Centrifuge at 500 x g for 5 minutes at 4°C. Remove the supernatant, leaving behind just enough to cover the pellet. Do not dry pellet.

15| Resuspend the pellet in 300 μL NWRB with 1% BSA and 3 μL of the DAPI stock (final concentration 10 μg / μL) by either inverting 5 times or pipetting up and down 5 times with a wide-bore tip.

To resuspend the pellet gently invert the sample several times. If it fails to resuspend a 1 mL wide-bore pipette tip can be used to gently pipette mix the sample up and down up to 10 times

Full resuspension of the pellet is not essential and, in our hands, still yields excess of 100,000 haploid nuclei. Excessive inverting or mixing may damage the integrity of the nuclei.

16| Filter the sample using a 40 μm Flowmi cell strainer into a 5 mL polypropylene tube which is kept on ice and in the dark.

Cell sorting and cytometer settings

Cytometric analysis should be performed as soon as possible; ideally within 10–30 minutes post-isolation. It is recommended to work with an experienced cytometric operator to optimise the sorting procedure under your laboratory’s conditions. We recommend using a 75 μm nozzle, and a flow rate of fewer than 10,000 events per second. It is critical to optimise your sort for purity rather than yield. Refer to your instrument manufacturers setup and operation guide.

Prior to nuclei sorting turn on the cytometer as per the manufacturer’s instructions. Use a collection device capable of holding 5 mL polypropylene tubes and ideally maintaining 4°C.

Diploid control material–here, the single splenic nuclei suspension–is used to identify the G1 diploid peak based on DAPI intensity, which in turn is used to identify haploid cells from reproductive tissue (Fig 2). This G1 peak–of the diploid sample–is placed in-range, visually, on the x-axis by adjusting the voltage. Haploid sperm nuclei can then be distinguished from diploid nuclei using the same gating parameter but with a final DAPI intensity approximately half that of the diploid control.

Fig 2

Flow cytometric gating strategy for the isolation of haploid cells.

The following gating steps are applied sequentially, as indicated by the arrows i.e. all events in ‘B’ represent only the gated events from ‘A’ and so on to ‘F’. (A) Nuclei are isolated from the rest of the population initially using linear forward scatter area (FSC-A-lin) and linear side scatter area (SSC-A-lin) by region gate 1 (R1). (B) Single nuclei are isolated using: region gate 2 (R2) on linear side scatter width (SSC-W-lin) and linear side scatter height (SSC-H-lin); (C) region gate 3 (R3) on linear forward scatter width (FSC-W-lin) and linear forward scatter height (FSC-H-lin); (D) region gate 4 (R4) on linear DAPI area (DAPI-A-lin) and linear DAPI width (DAPI-W-lin); and (E) region gate 5 (R5) on linear DAPI area (DAPI-A-lin) and linear DAPI height (DAPI-H-lin) (F) The use of a diploid control allows flow cytometric gating of haploid sperm. Diploid samples such as splenic cells used here, will have a G1 (2c DNA content) and a G2 peak (4c DNA content, out of range in this figure) peak respectively. Acquire events from region 6 (R6), the haploid nuclei which have a 1c DNA content (R6) which are not present in the diploid control sample. The values on all both axes of panels A-E and the x-axis of panel F are arbitrary values that represent increasing intensity of signal from 0, the lowest, to 250K the greatest. Major and minor ticks represent 50K and 10K increments respectively.

17| Preparation of FACS collection tubes and plates–For each sample add 200 μL of NWRB with 0.1% BSA in a 5 mL polypropylene tube. For collections in 96-well plates add 50 μL of NWRB with 0.1% BSA.

18| Critical step–Set the sorter precision to ‘4-Way Purity’, this will ensure that the final volume of the sorted nuclei is kept to a minimum (~1 nucleus sorted per nL), preventing dilution of the sample with FACS sheath solution.

On some instruments this setting may be called ‘Single Cell’ precision when sorting in 96-well plates.

It is preferable to use slow flow rates, but a medium flow rate can be used if necessary.

19| Using the standard workflows of your FACS facility, gate for individual nuclei (Fig 2A–2E). Gate the main population of nuclei using linear forward scatter area (FSC-A-lin) and linear side scatter area (SSC-A-lin) by region gate 1 (R1) (Fig 2A). Then gate single cells R2, R3, R4, R5 (Fig 2B–2E).

20| To discriminate haploid from diploid nuclei (R6), use the DAPI stain to quantify the DNA content of each nucleus (Fig 2F). Splenic nuclei serve as diploid controls here, which are essential to confidently gate around the peak that corresponds to the DAPI-stained haploid nuclei (Fig 2F). It is therefore important to set the voltage of the laser that excites DAPI allow visualisation of the G1 peak of the diploid sample, as well as a haploid peak, in the same range on the x-axis (Fig 2F, right). The haploid peak–on the x-axis of a histogram–will be half that of the diploid G1 peak.

21| Diploid nuclei control sorting–Sort the target number of diploid nuclei–typically 100,000 or greater–into the previously prepared 5 mL polypropylene tube or 96 well plate.

While this diploid control sample is generally not used for library preparation and sequencing, it is helpful to perform sorting of individual nuclei from this sample as it is typically easier to isolate 100,000 splenic nuclei than 100,000 haploid nuclei from testis while establishing the protocol.

22| Haploid sample sorting–Sort the target number of haploid nuclei–typically 100,000 in our experiments–into the previously prepared 5 mL polypropylene tube or 96 well plate.

The time required to isolate 100,000 cells is dependent on the concentration of nuclei in solution, but typically splenic and testicular samples require 2 minutes to reach this target.

If material is very limiting, it might be helpful to consider a plate-based method to maximise yield and therefore to sort one nucleus per well of an appropriate plate at this step.

23| Centrifuge the samples at 500 x g for 5 min at 4°C. Remove the supernatant, leaving approximately 50 μL. Do not allow the pellet to dry.

Post-FACS concentration of nuclei (for droplet-based methods)

At this point the nuclei can be used for a variety of applications, including single- and bulk-nuclei sequencing.

The specific application will determine what buffer the nuclei should be exchanged into. Below we provide a case study where we isolated haploid sperm nuclei for library preparation with the droplet-based 10x Genomics single-nucleus ATAC-seq kit.

24| Add 300 μL NWRB with 0.1% BSA and 0.01% Digitonin and incubate on ice for 5 minutes.

25| Centrifuge at 500 x g for 5 min at 4°C. Remove the supernatant, leaving behind ~50 μL. Do not dry the pellet.

26| Add 300 μL Dilute Nuclei Buffer (10x Genomics) and gently resuspend the nuclei by pipetting up and down 3 times using a wide-bore pipette tip.

27| Centrifuge at 500 x g for 5 min at 4°C. Remove the supernatant, leaving behind ~50 μL. Do not allow the pellet to dry.

28| Gently resuspend the nuclei by pipetting up and down 3 times using a wide-bore pipette tip.

29| Quantify the concentration of the nuclei suspension using a hemocytometer. We use a Leica Thunder under 40x magnification, with brightfield to locate the hemocytometer grid and fluorescence to identify the DAPI-stained nuclei. This also allows use to check the integrity of the nuclei (Fig 3).

Fig 3

Intact haploid sperm and spermatids are used as input for droplet-based or plate-based high-throughput sequencing library construction.

Representative image of high-quality intact haploid cells isolated using the protocol.

For droplet-based methods of partitioning single nuclei, such as the 10x single-nucleus kits, all nuclei must be intact.

We have used this method and reliably achieved a final concentration of 400 to 450 nuclei per μL, with the aim of loading 1500 nuclei into a 10x Chromium.

30| Follow the manufacturer’s protocol for library preparation. From here we have made high-quality “single-nucleus” and “bulk” libraries with the haploid nuclei with the Next GEM Single Cell ATAC Library Kit v1.1 (10x Genomics) and with Nextera (Illumina) DNA Library Prep Kit respectively. For bulk sequencing we aim to sequence 50,000 nuclei.

31| The sequencing depth required per cell will vary according to experimental aims. We have found in the early stages of protocol development that a useful guide can be to sequence a given sample in two steps. First, request your sequencing platform obtain approximately 200 million paired-end reads per one thousand expected cells. Once this data has been analysed, and cell number and coverage is approximated, a second round of sequencing is performed to obtain the required depth per cell.

If the recovery of total number of nuclei recovered is lower than expected, in the first instance it is important to independently assess how many cells are present by assessing the number of barcodes with > 800,000 high-quality paired-end reads that map to the target genome. From here the threshold can be adjusted based on the researchers’ decisions.

Next time the entire experiment is performed, the number of nuclei used for library preparation can be doubled as nuclei concentration should not be limiting.

When using bulk- or single-nucleus ATAC-seq, the nuclei permeabilization step with digitonin, or similar detergents, and the associated buffers can be re-optimised.

Sequencing and bioinformatics considerations

The protocol described in this peer-reviewed article is published on protocols.io (dx.doi.org/10.17504/protocols.io.6qpvr67jbvmk/v1) and is included for printing purposes as S1 File.

Variations of the protocol for other applications

The protocol can be adapted to work with plate-based methods. Steps 1–21 should provide a sensible starting point to isolate high-quality haploid nuclei. Depending on the desired library preparation method (e.g. ATAC or whole-genome amplification) user-specific modifications can be made at the appropriate steps.

Troubleshooting

If poor yields are obtained or nuclei do not appear intact, we suggest optimising the salt concentrations of the PBS. The tonicity that is ideal for mouse and human cells can differ and may be the cause of cases of cytolysis.

Further, the visualisation of cells and nuclei at every stage is possible with bright field microscopy or immunofluorescence with DAPI. Visualisation of sample integrity and concentration should be used routinely to establish the protocol, and when problems occur, to identify at which stage cells or nuclei are lost or perturbed.

Anticipated results

The anticipated results with the haploid sperm nuclei isolation protocol depend on the mouse genetic background and age. The protocol was developed with wild-type mice and mice with mutations in genes required for spermatogenesis may affect the yield of this protocol. The successful procedure is dependent on working proficiently with the mice and their biological material, the composition of reagents, the materials and proficient use of the cell sorter. Upon visualisation of DAPI-stained haploid nuclei a distinct sperm head shape should be seen. No extruded chromosomes should be visible as this will result in cross contamination between cells when analysing sequencing results.

Homogenisation of splenic cells as diploid controls.

Cartoon representation of how splenic cells are obtained via homogenisation and filtration.

(TIFF)

Click here for additional data file.

Yields of intact single haploid nuclei with the protocol.

Yield data from our laboratory using the protocol since optimisation. The data is from fresh and frozen testes, fresh epididymides, and cover wild-type and hypospermatogenic (Fancm-deficient) mice. “gated haploid nuclei” represents the total number of haploid nuclei that were gated using the strategy shown in Fig 2A–2F and isolated at gate “R6” (Fig 2F). “haploid percent of population (events pre-gating)” represents the number of haploid nuclei were gated (R6, Fig 2F) for a given experiment divided by the total number of events detected pre-gating. i.e. (R6 / events pre-gating) X 100.

(XLSX)

Click here for additional data file.

Step-by-step protocol, also available on protocols.io.

(PDF)

Click here for additional data file.

12 Jul 2022

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Additional Editor Comments:

Both expert reviewers in the field found the protocol interesting and that it could be a good addition to the panel of available techniques. However, they both ask for several clarifications that need to be considered. Please revise accordingly.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Does the manuscript report a protocol which is of utility to the research community and adds value to the published literature?

Reviewer #1: Yes

Reviewer #2: Yes

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2. Has the protocol been described in sufficient detail?

Descriptions of methods and reagents contained in the step-by-step protocol should be reported in sufficient detail for another researcher to reproduce all experiments and analyses. The protocol should describe the appropriate controls, sample sizes and replication needed to ensure that the data are robust and reproducible.

Reviewer #1: No

Reviewer #2: Yes

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3. Does the protocol describe a validated method?

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Reviewer #1: Yes

Reviewer #2: No

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Reviewer #1: N/A

Reviewer #2: No

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Reviewer #1: Yes

Reviewer #2: No: It is a clear and concise description, I noticed only some minor typos:

line 127 – placed

lines 151/152 – squeezing the testis with pointed tweezers until the testis bursts

line 156 – microcentrifuge tube

line 192 – Not a functional sentence

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6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The paper by Novakovic et al present a method for the isolation of haploid nuclei from fresh and frozen mice testis. The methods can be applied for several purposes as indicated by the Authors, who also anticipate that the method could be used also for human specimens.

The method is of interest, however there are some points needing detailing and clarification.

Specific points:

1. The Authors do not report any data on the yield either of the global method or of the cell sorting step, whereas it appears an important detail. It appears that a huge number of haploid nuclei is lost as the method uses one testis and recoveries 100,000 (or 300,000?, line 185). Thus this method could be used for only certain applications. Have the Authors any explanation for such high cell lost? This point is important because if the method used human specimens, it is expected that the available material would be much smaller and the question is whether the starting material was sufficient with this very low yield.

2. Cell sorting. Details on strategy to sort haploid nuclei is completely lacking. Which gate in FSC/SSC dot plot? Did you exclude doublets? Which were the technical settings of the instrumentation?

3. Line 213. The haploid peak is represented in a fluorescence distribution histogram. What do you mean with density plot? In addition, use “gate around peak” not “gate with”.

4. In the abstract, the Author claim that the method is reproducible, but non data on this point are provided

Minor:

Figure 1 is confused with Figure 2

Referring to real Figure 1 is lacking in the text

Line 195, G1 peak does not seem centred on x-axis.

Reviewer #2: The protocol provides a valuable addition to the field of single cell assays of testis tissue, as especially the nucleus isolation has proven tricky in the past. The description of working steps is concise, and I would feel confident following this protocol.

However, I do have some concerns with the manuscript:

1) The authors refer to the method as “single-sperm nucleus isolation”. I find this misleading. Mouse testis contains haploid spermatids at different stages in development and transcription profiles differ distinguishably from round spermatids to later spermatid stages. The nucleus shown in figure 2C corresponds to a later spermatid stage, however FACS sorting of the haploid population would in my opinion also include more immature round spermatids. If this population is excluded in the protocol, it is unclear how. This should be discussed and Figure 2 should be updated to depict not only a single nucleus, but a representative sample of the obtained haploid nuclei.

2) In line with my first concern, the authors imply that this protocol can be adapted to isolate mature human sperm. Maturation of sperm, including development of fertilisation capability and motility, is completed not in the testis, but in the epididymides. In my experience, motile sperm is “more sticky” than spermatids and it would be interesting to see how the protocol performs with this population. At least, the distinction between spermatids, spermatozoa and mature sperm should be made clear in the manuscript and discussed in more detail.

3) I was unable to assess the data from the case study, as it was not made available to reviewers prior to publication. Thus, I feel a bit uncomfortable to classify this as a validated method. At least the quality control data for the case study, for example an image of isolated nuclei in the hemocytometer to assess nuclear integrity, should be included.

In light of these points, I recommend the manuscript be revised.

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29 Jul 2022

Our responses are pasted below. However, we have attached this response to reviewers as a word document, which contains formatting which we believe will improves the readability of the responses.

Dear Dr Drevet

We thank you and the reviewers for your thoughtful reading of our manuscript and your helpful comments. The reviewers are complimentary on the quality and value of the protocol e.g. both reviewers confirmed that the protocol “is of utility to the research community and adds value to the published literature”, and “The protocol provides a valuable addition to the field of single cell assays of testis tissue, as especially the nucleus isolation has proven tricky in the past. The description of working steps is concise, and I would feel confident following this protocol.” We address their questions, and all suggestions, below.

*Please note that references to line numbers in the manuscript are for the clean (without tracked changes) version of the revised manuscript.

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WC and DJM received funding related to this work from the Australian National Health and Medical Research Council (GNT1129757, GNT1185387).

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WC and DJM received funding related to this work from the Australian National Health and Medical Research Council (GNT1129757, GNT1185387).

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The full ethics statement is now included on lines 140-141 and reads:

“All experimental procedures were approved in writing by the St. Vincent’s Hospital Melbourne Animal Ethics Committee.”

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Reviewers' comments:

Reviewer #1:

The paper by Novakovic et al present a method for the isolation of haploid nuclei from fresh and frozen mice testis. The methods can be applied for several purposes as indicated by the Authors, who also anticipate that the method could be used also for human specimens.

The method is of interest, however there are some points needing detailing and clarification.

Specific points:

1. The Authors do not report any data on the yield either of the global method or of the cell sorting step, whereas it appears an important detail. It appears that a huge number of haploid nuclei is lost as the method uses one testis and recoveries 100,000 (or 300,000?, line 185). Thus this method could be used for only certain applications. Have the Authors any explanation for such high cell lost? This point is important because if the method used human specimens, it is expected that the available material would be much smaller and the question is whether the starting material was sufficient with this very low yield.

We appreciate that this warrants clarification, both in the manuscript and here in this response. We have included a new Supplementary Table 1 which has data for all experiments that used this technique since it was optimised. We have also referenced a new manuscript which is on Biorxiv and under review at PNAS, which has used this technique to identify reproducible phenotypic differences between genotypes (Tsui et al, Figure 1b, https://doi.org/10.1101/2022.06.16.496499).

From a practical perspective, the protocol has been optimised for efficiency – less than an hour – and stringent quality of intact nuclei as opposed to yield. We typically set a target number of haploid cells and stop sorting once that has been reached. E.g. in a very simple example if a user wanted to use a plate-based library preparation approach, and the researcher were using a 384-well plate, they would only require 384 nuclei; one in each well. With respect to numbers such as “100,000” frequently mentioned in the original submission, this stems from aiming to obtain sequencing data for 1,000 nuclei from a droplet-based method (e.g. with 10x Genomics library preparation kits). And to prepare the sequencing libraries with an appropriate concentration of nuclei, based on constraints set by these kits and “droplet encapsulation rates”, we aim to sort 100,000 haploid cells. We have aimed to not place excessive focus on 10x kits as the intended downstream application of this method, but we appreciate that further clarification of where the target number of nuclei originates is required.

We clarify on lines 61-63;

- “We use 100,000 as a minimum target number in this protocol as droplet-based single-nucleus library preparation methods can require a high concentration of nuclei in a small volume.”

and lines 307-310.

- “For droplet-based methods of partitioning single nuclei, such as the 10x single-nucleus kits, all nuclei must be intact. We have used this method and reliably achieved a final concentration of 400 to 450 nuclei per μL, with the aim of loading 1500 nuclei into a 10x Chromium.”

From a technical perspective, using this protocol, we can routinely sort more than 300,000 haploid nuclei from a single mouse testis. However, due to our strict selection criteria for single cells (on FACS), approximately 50% of the total sorted nuclei are gated out. As our intention is to sequence individual nuclei, often with droplet-based library methods which have steps where all nuclei are in solution adjacent to one another from protocol steps 23-30 inclusive, it is essential that all nuclei included are intact. If one nucleus included is not intact and extrudes its contents into solution, it can contaminate the data of an unknown quantity of other nuclei when they are encapsulated with the “unwanted” free-floating nucleic acid. This need for the integrity of all nuclei is referred to on lines 307-308 and reads:

“For droplet-based methods of partitioning single nuclei, such as the 10x single-nucleus kits, all nuclei must be intact.”

If starting material is very low, a user can consult the newly added Supplementary Table 1 to consider this protocol could be suitable for their needs. Also, sorting individual nuclei into a plate is probably more appropriate in that situation. This comment is added to step 22 and now reads:

“If material is very limiting, it might be helpful to consider a plate-based method to maximise yield and therefore to sort one nucleus per well of an appropriate plate at this step.”

2. Cell sorting. Details on strategy to sort haploid nuclei is completely lacking. Which gate in FSC/SSC dot plot? Did you exclude doublets? Which were the technical settings of the instrumentation?

We have included a new Figure 2, which illustrates the gating strategy. The gating strategy and technical settings of the instrumentation have been expanded on in in the section “Cell sorting and cytometer settings”, from lines 212-271 and in particular in steps 19-20. This now reads:

Step 19. “Using the standard workflows of your FACS facility, gate for individual nuclei (Figure 2a-e). Gate the main population of nuclei using linear forward scatter area (FSC-A-lin) and linear side scatter area (SSC-A-lin) by region gate 1 (R1) (Figure 2a). Then gate single cells R2, R3, R4, R5 (Figure 2b-e).”

Step 20. “To discriminate haploid from diploid nuclei (R6), use the DAPI stain to quantify the DNA content of each nucleus (Figure 2f). Splenic nuclei serve as diploid controls here, which are essential to confidently gate around the peak that corresponds to the DAPI-stained haploid nuclei (Figure 2f). It is therefore important to set the voltage of the laser that excites DAPI allow visualisation of the G1 peak of the diploid sample, as well as a haploid peak, in the same range on the x-axis (Figure 2f, right). The haploid peak – on the x-axis of a histogram – will be half that of the diploid G1 peak.”

3. Line 213. The haploid peak is represented in a fluorescence distribution histogram. What do you mean with density plot? In addition, use “gate around peak” not “gate with”.

We incorrectly used the term “density plot” where we should have used “histogram”. This has been corrected, and now reads:

“The haploid peak – on the x-axis of a histogram – will be half that of the diploid G1 peak.” [lines 254-255]

We clarified the wording about gates as appropriate too. This now reads:

“Splenic nuclei serve as diploid controls here, which are essential to confidently gate around the peak that corresponds to the DAPI-stained haploid nuclei (Figure 2f)” [lines 250-252].

4. In the abstract, the Author claim that the method is reproducible, but non data on this point are provided

We have changed the word “reproducible” to “reliable”. We appreciate the word “reproducible” conjures up an expectation of “reproducible phenotypes”. Whereas we mean a “reliable protocol” that works every time, assuming other aspects of quality assurance in the user’s laboratories are met.

We have included a new Supplementary Table 1 which has data for all experiments that used this technique since it was optimised.

Minor:

Figure 1 is confused with Figure 2

We now refer, accurately, to Figure 2 [line 224].

Referring to real Figure 1 is lacking in the text

We now refer to Figure 1 in a number of appropriate sections [lines 61, 77, 144, 177].

Line 195, G1 peak does not seem centred on x-axis.

We modified the text to instruct that the G1 peak needs to be “in range”. This sentence on now reads;

“This G1 peak – of the diploid sample – is placed in-range, visually, on the x-axis by adjusting the voltage.” [lines 224-225]

Reviewer #2:

The protocol provides a valuable addition to the field of single cell assays of testis tissue, as especially the nucleus isolation has proven tricky in the past. The description of working steps is concise, and I would feel confident following this protocol.

However, I do have some concerns with the manuscript:

1) The authors refer to the method as “single-sperm nucleus isolation”. I find this misleading. Mouse testis contains haploid spermatids at different stages in development and transcription profiles differ distinguishably from round spermatids to later spermatid stages. The nucleus shown in figure 2C corresponds to a later spermatid stage, however FACS sorting of the haploid population would in my opinion also include more immature round spermatids. If this population is excluded in the protocol, it is unclear how. This should be discussed and Figure 2 should be updated to depict not only a single nucleus, but a representative sample of the obtained haploid nuclei.

We have modified the text and a new Figure 3 to illustrate that this protocol is designed to isolate all types of haploid cells from a male, that are available from the given tissue; from round spermatids to mature sperm. We also make clear in the text that the protocol is designed to isolation both spermatids and spermatozoa:

“This method is developed to isolate nuclei from both spermatids and spermatozoa.” [line 63]

2) In line with my first concern, the authors imply that this protocol can be adapted to isolate mature human sperm. Maturation of sperm, including development of fertilisation capability and motility, is completed not in the testis, but in the epididymides. In my experience, motile sperm is “more sticky” than spermatids and it would be interesting to see how the protocol performs with this population. At least, the distinction between spermatids, spermatozoa and mature sperm should be made clear in the manuscript and discussed in more detail.

We agree that we did not make sufficiently clear that our motivation for developing this protocol was due to our own technical challenges due to the stickiness of these cell types. Therefore, we developed a method that focuses on the isolation of intact nuclei. This is now made explicit and reads:

“A major challenge for the isolation of individual haploid cells from males for sequencing is that they stick to most pipette tips and tubes, and traditional methods for bulk sperm isolation use technically challenging density gradients. Therefore, the motivation for developing this technique is to have a simple and rapid nucleic acid preparation technique that is compatible with current sequencing technologies,…” [lines 42-46].

We also have added new data in Supplementary Table 1, showing how with epididymides we used the protocol to isolate between 320,000 to 1,100,000 haploid nuclei.

3) I was unable to assess the data from the case study, as it was not made available to reviewers prior to publication. Thus, I feel a bit uncomfortable to classify this as a validated method. At least the quality control data for the case study, for example an image of isolated nuclei in the hemocytometer to assess nuclear integrity, should be included.

In light of these points, I recommend the manuscript be revised.

We have included a new Supplementary Table 1 which has data for all experiments that used this technique since it was optimised. We have modified Figure 2 as suggested to show more examples of nuclear integrity. We have also referenced a new manuscript which is on Biorxiv and under review at PNAS, which has used this technique to identify reproducible phenotypic differences between genotypes

(Tsui et al, Figure 1b, https://doi.org/10.1101/2022.06.16.496499).

Minor

(taken from the reviewers’ field: “Is the article presented in an intelligible fashion and written in standard English?”)

Reviewer #1: Yes

Reviewer #2: No: It is a clear and concise description, I noticed only some minor typos:

line 127 – placed

Done [line 151]

lines 151/152 – squeezing the testis with pointed tweezers until the testis bursts

Done [lines 176-177]

line 156 – microcentrifuge tube

Done [lines 183-184]

line 192 – Not a functional sentence

We modified this sentence for clarity, to read;

Diploid control material – here, the single splenic nuclei suspension – is used to identify the G1 diploid peak based on DAPI intensity, which in turn is used to identify haploid cells from reproductive tissue (Figure 2f). [lines 228-230]

Submitted filename: Response to Reviewers.docx

Click here for additional data file.

30 Aug 2022

1 Sep 2022

Dear Dr Drevet

Thank you for sharing reviewer #1’s comments. Reviewer #1 acknowledges that we made the requested improvements. They raise two minor points which are incorporated into this revised version of the manuscript and are described concisely below.

(please see the attached word document for the same responses as below, but with better formatting)

Reviewer #1: The Authors amended the MS according to requests. However, I have some further questions on the newly added material.

Supplemental figure 1. Please better explain: in the “nuclei haploid events” column, there is the final number of aploid nuclei, i.e. that post sorting. In the last column (percentage haploid of population), are the percentages referred to what? To the total haploid nuclei? To the total acquired haploid nuclei? For instance, in the first row, 100,000 nuclei represent the 26,7% of what? How did you count the total haploid nuclei?

Response - We have expanded the legend of S1 Table to explain what these terms represent. Further we have improved the terms used. We have redefined “nuclei haploid events” to be “gated haploid nuclei”. Similarly, “percentage haploid of population” was changed to “haploid percent of all events pre-gating”

The new S1 Table legend now reads:

“S1 Table. Yields of intact single haploid nuclei with the protocol:

Yield data from our laboratory using the protocol since optimisation. The data is from fresh and frozen testes, fresh epididymides, and cover wild-type and hypospermatogenic (Fancm-deficient) mice. “gated haploid nuclei” represents the total number of haploid nuclei that were gated using the strategy shown in Fig 2A-F and isolated at gate “R6” (Fig 2F) . “haploid percent of all events pre-gating” represents the number of haploid nuclei were gated (R6, Fig 2F) for a given experiment divided by the total number of events detected pre-gating. i.e. (R6 / events pre-gating) X 100.”

Reviwer #1 - In figure 2, the titles of the axes are not readable. Similarly for values in the axes (too small characters). In addition, it is not clear whether the gates were applied one within the other (from A to E) or not (i.e. independently of each other). In particular, the histograms of figures F from which dot plot are generated? This information has to be added.

Response - We agree and we have made these suggested changes. We have shown that the gating strategy from Fig 2A to 2F was applied sequentially to gated events from the previous step, both on the figure with arrows, and specified in the figure legend. Further, the axis titles have been increased as requested, as have the numbers on the axes. As the intensity numbers (0-250K) are arbitrary we removed the numbers from 50K-200K to create more space for larger font for the minimum and maximum values, but explain in the Fig 2 legend the scale that the major and minor ticks represent 50K and 10K respectively. We also upload an electronic vectorized version of the image that will ensure high resolution can be rendered into the published version.

The new Figure 2 legend now reads:

“Fig 2. Flow cytometric gating strategy for the isolation of haploid cells.

12 Sep 2022

SSNIP-seq: A simple and rapid method for isolation of single-sperm nucleic acid for high-throughput sequencing

PONE-D-22-14844R2

Dear Dr. W.  Crismani,

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19 Sep 2022

PONE-D-22-14844R2

SSNIP-seq: A simple and rapid method for isolation of single-sperm nucleic acid for high-throughput sequencing

Dear Dr. Crismani:

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on behalf of

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