Differential expression analysis of transcriptome data of Trypanosoma brucei RBP6 induction in procyclics leading to infectious metacyclics and bloodstream forms in vitro.

We used an in vitro system based on the inducible expression of the RNA binding protein 6 (RBP6) to monitor transcriptome changes during the differentiation of Trypanosoma brucei from non-infectious procyclics to infectious metacyclics and from metacyclics to bloodstream forms. This data file describes the bioinformatics analysis of 20 distinct RNA-Seq samples, with four biological replicates each, highlighting differential transcript abundance. Additional functional annotation analysis using Gene Ontology is also presented. Complete raw data files were deposited at the NCBI Sequence Read Archive - SRA at http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi with accession numbers: SRP153824, SRP153562, and SRP152737.

Specifications Table

Value of the data

This is the first transcriptome analysis of Trypanosoma brucei differentiation from procyclics to metacyclics and from metacyclics to bloodstream forms.

The differential expression analysis provides a data source for the understanding of how Trypanosoma brucei becomes infectious.

The transcriptome changes might in the future lead to a better understanding how metacyclic to bloodstream form differentiation is orchestrated.

Data

This data consists of 20 high-throughput sequencing samples with four biological replicas each of wild-type RBP6 and mutant RBP6 (Q109K) induced for 6 and 5 days, respectively. In addition, metacyclics from the mutant RBP6 induction were transformed into bloodstream forms over an 11-day period. Raw data is available through NCBI׳s Sequence Read Archive through the direct link http://www.ncbi.nlm.nih.gov/sra? with accession numbers: SRP153824, SRP153562, and SRP152737.

Experimental design, materials, and methods

Experimental design

RBP6 (Tb927.3.2930) was inducibly expressed in the Trypanosoma brucei Lister 427(29-13) procyclic strain as described [1], [2]. Metacyclics were purified at room temperature on zirconia/silica beads columns [3]. Purified metacyclics were cultured in bloodstream form conditions at 37 °C.

Total RNA was prepared from 2 × 107 procyclics, purified metacyclics, and bloodstream form cells with the TRIzol reagent (Invitrogen) according to the manufacturer׳s instructions. Four independent samples, i.e., biological replicas, were prepared from each life cycle stage and from each time point in a time course and isolation of poly(A)+ mRNA, library preparation and sequencing on an Illumina HiSeq. 2500 platform were performed at the Yale Center for Genome Analysis. 75 nt long reads were mapped to the T. brucei 11 megabase chromosomes (GeneDB version 5) using the Lasergene 14.1.2 software package from DNASTAR as described [1]. Reads assigned per kilobase of target per million mapped reads (RPKM) was used for normalization and only open reading frames were used for the calculation of RPKM values. Differential gene expression analysis was done with the Lasergene software package from DNASTAR [1] and the Gene Ontology (GO) enrichment tool on the TriTrypDB webserver (http://tritrypdb.org/) was used to analyze the differentially expressed genes for functional annotation and GO terms were condensed by submitting to REVIGO [4].

Subject area	Biology
More specific subject area	Molecular Parasitology
Type of data	Tables and raw data
How data was acquired	High-throughput sequencing carried out by The Yale Center for Genome Analysis (YCGA) using an Illumina HiSeq 2500.
Data format	Raw, analyzed
Experimental factors	RNA isolation, cDNA library construction and sequencing
Experimental features	Total RNA was extracted from time course samples. Library preparation and sequencing was performed by YCGA. Analysis was carried out in-house.
Data source location	New Haven, Connecticut, USA
Data accessibility	RNA-Seq data from this study have been submitted to the NCBI Sequence Read Archive – SRA at http://www.ncbi.nlm.nih.gov/Traces/sra/sra.cgi with accession numbers: SRP153824, SRP153562, and SRP152737.
Related research article	A single-point mutation in the RNA-binding protein 6 generates Trypanosoma brucei metacyclics that are able to progress to bloodstream forms in vitro
	Huafang Shi, Kiantra Butler1 and Christian Tschudi*
	Department of Epidemiology of Microbial Diseases, Yale School of Public Health, 60 College Street, New Haven, Connecticut 06520, USA.
	1 Present address: Division of Scientific Resources, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, 1600 Clifton Road, Atlanta, GA 30329, USA.
	*Corresponding author at: Department of Epidemiology of Microbial Diseases, Yale School of Public Health, Boyer Center for Molecular Medicine 136C, 295 Congress Avenue, New Haven, Connecticut 06536, USA. Tel.: +1 203 785 7332. E-mail: christian.tschudi@yale.edu
	Molecular and Biochemical Parasitology, 224 (2018) 50-56.