Illumina Genome Analyzer IIx sequencing; GSM1169395: paTh1_4h_r1; Mus musculus; RNA-Seq

paTh1_4h_r1

Although lincRNAs are implicated in regulating gene expression in various tissues, little is known about lincRNA transcriptomes in the T cell lineages. Here we identify 1,524 lincRNAs in 42 T cell samples from early T cell progenitors to terminally differentiated T helper subsets. Our analysis revealed highly dynamic and cell-specific expression patterns of lincRNAs during T cell differentiation. Importantly, these lincRNAs are located in genomic regions enriched for protein-coding genes with immune-regulatory functions.  Many of these transcripts are bound and regulated by the key T cell transcription factors, T-bet, GATA3, STAT4 and STAT6. We demonstrate that the lincRNA LincR-Ccr2-5''AS, together with GATA3, is an essential component of a regulatory circuit in Th2-specific gene expression. Overall design: To obtain comprehensive profiles of lincRNA expression during the development and differentiation of T cell lineages, we purified CD4-CD8 double negative (DN) cells (DN1, DN2, DN3 and DN4), double positive (DP) cells (CD4+CD8+CD3low and CD4+CD8intCD69+), single positive (SP) CD4 and CD8 cells, and thymic-derived regulatory T cells (tTreg) from thymi of C57BL/6 mice. Additionally, we obtained Th1, Th2, Th17 and iTreg cells by in vitro differentiation of naïve CD4 T cells for a various length of time in culture (4 hrs, 8 hrs, 12 hrs, 24 hrs, 48 hrs, 72 hrs, 1 week, 2weeks). Total and/or polyadenylated RNAs from these cells was analyzed using RNA-Seq. To understand the regulation of lincRNAs by T cell master regulator T-bet, we compared the transcriptiomes between T-bet deficient Th1 cells and control Th1 cells. We did similar experiments and data analysis for STAT4 (Th1), GATA3 (Th2) and STAT6 (Th2). Finally, to address the funcation of a Th2-specifically expressed lincRNA, lincR-Ccr2-5''AS, we compared the transcriptomes between lincR-Ccr2-5''AS knockdown Th2 cells and control Th2 cells.

Expression and regulation of lincRNAs during T cell development and differentiation

Submitted by Gene Expression Omnibus on 11-NOV-2013

Although lincRNAs are implicated in regulating gene expression in various tissues, little is known about lincRNA transcriptomes in the T cell lineages. Here we identify 1,524 lincRNAs in 42 T cell samples from early T cell progenitors to terminally differentiated T helper subsets. Our analysis revealed highly dynamic and cell-specific expression patterns of lincRNAs during T cell differentiation. Importantly, these lincRNAs are located in genomic regions enriched for protein-coding genes with immune-regulatory functions.  Many of these transcripts are bound and regulated by the key T cell transcription factors, T-bet, GATA3, STAT4 and STAT6. We demonstrate that the lincRNA LincR-Ccr2-5''AS, together with GATA3, is an essential component of a regulatory circuit in Th2-specific gene expression. Overall design: To obtain comprehensive profiles of lincRNA expression during the development and differentiation of T cell lineages, we purified CD4-CD8 double negative (DN) cells (DN1, DN2, DN3 and DN4), double positive (DP) cells (CD4+CD8+CD3low and CD4+CD8intCD69+), single positive (SP) CD4 and CD8 cells, and thymic-derived regulatory T cells (tTreg) from thymi of C57BL/6 mice. Additionally, we obtained Th1, Th2, Th17 and iTreg cells by in vitro differentiation of naÃ¯ve CD4 T cells for a various length of time in culture (4 hrs, 8 hrs, 12 hrs, 24 hrs, 48 hrs, 72 hrs, 1 week, 2weeks). Total and/or polyadenylated RNAs from these cells was analyzed using RNA-Seq. To understand the regulation of lincRNAs by T cell master regulator T-bet, we compared the transcriptiomes between T-bet deficient Th1 cells and control Th1 cells. We did similar experiments and data analysis for STAT4 (Th1), GATA3 (Th2) and STAT6 (Th2). Finally, to address the funcation of a Th2-specifically expressed lincRNA, lincR-Ccr2-5''AS, we compared the transcriptomes between lincR-Ccr2-5''AS knockdown Th2 cells and control Th2 cells.

For sequencing total RNA: the total RNAs were purified using Qiagen's miRNeasy micro kit (Qiagen cat# 217084) plus Qiagen’s DNase set (Qiagen cat# 79254) for on-column DNase digestion option. For sequencing PolyA RNA: PolyA-tailed RNAs were purified from pure total RNA using Dynabeads® mRNA DIRECT™ Kit (invitrogen cat# 610.12). Both were performed according to the kits' user manuals. For STAT4 and STAT6 KO: total RNAs were isolated using mirVana (Applied Biosystems) isolation kit. RNA concentration was determined using the Qubit RNA broad range assay in the Qubit Fluorometer (Invitrogen) and RNA integrity was determined using Eukaryote Total RNA Nano Series II ChIP on a 2100 Bioanalyzer (Agilent). Three independent biological replicates were pooled for RNA-seq. For total RNA-seq library prep: 10ng of purified total RNA was used for cDNA amplification using the Ovation RNA-Seq System V2 (NuGEN Technologies, Inc. cat# 7102-08). then 200ng of ds cDNA was sonicated in Diagenode's Bioruptor (level M, for a  total of 30 minutes of 20 seconds ON and 20 seconds OFF) to a size range of 100-400bp. For mRNA-seq library prep:  a random primer (invitrogen cat# 48190-011) was used to prime the purified PolyA tailed-RNA and the cDNA synthesis was performed using SuperScript® Double-Stranded cDNA Synthesis Kit (Invitrogen cat# 11917-010)  according to the user’s manual. The ds cDNA was subject to sonication in Diagenode's Bioruptor (level M, total of 30 minutes of 20 seconds ON and 20 seconds OFF). Indexed library was prepared using Illumina’s multiplexing sample prep oligonucleotide kit (Ref# 1005709) with Epicentre's End-It DNA End-repair Kit (Epcentre, cat# ER81050) according to the user's manual and Illumina's multiplexing sample preparation guide. For STAT4 and STAT6 KO: RNA-seq libraries were prepared from 4 ug of total RNA using the TruSeq RNA sample prep kit following manufacturer’s protocol (Illumina). Briefly, oligo-dT purified mRNA was fragmented and subjected to first and second strand cDNA synthesis. cDNA fragments were blunt-ended, ligated to Illumina adaptors, and PCR amplified to enrich for the fragments ligated to adaptors.

Expression and regulation of lincRNAs during T cell development and differentiation

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