RNA-Seq of mus musculus WT C57Bl/6-Ly5.2 DN3b cells from Thymus
RNA from sorted DN3b cells (Lin-CD25+CD44-CD27+) from Tcf1-/- and wild type littermates thymi was isolated using the Mini RNeasy Kit (Qiagen) The integrity (scores > 9.0) of the RNA was determined on the Agilent 2100 Bioanalyzer (Agilent). Total RNA enrichment for sequencing poly(A) RNAs was performed with the TruSeq mRNA sample preparation kit (Illumina). 1g of total RNA for each sample was used for poly(A) RNA selection using magnetic beads coated with poly-dT, followed by thermal fragmentation. The fragmented poly(A) RNA enriched samples were subjected to cDNA synthesis using Illumina TruSeq preparation kit. cDNA was synthesized by reverse transcriptase (Super-Script II) using poly-dT and random hexamer primers. The cDNA fragments were then blunt-ended through an end-repair reaction, followed by dA-tailing. Subsequently, specific double-stranded bar-coded adapters were ligated and library amplification for 15 cycles was performed. The pooled cDNA library consisted of equal concentration bar-coded samples. The pooled library was sequenced in one lane, 36bp single read on the HiSeq2500 (Illumina).