63_72Normal-Rep2
Total RNA was isolated from CD4+ T cells using TRIzol and performed by RNeasy Mini Kit. RNA concentration was assessed using Nanodrop ND-1000 spectrophotometer and RNA quality was determined by 2100 Bioanalyzer, only samples with RIN scores over 7 could be submitted for sequencing. Then mRNA was used to synthesize the first and the second strand cDNA by using SuperScriptTM III Reverse Transcriptase and oligo (dT) 12-18 primers. After purification, the double-strand cDNA (dscDNA) was fragmented into ~300 bp. The library construction was subsequently performed as follow. End repair of the fragmented dscDNA was performed and then 3 poly-A was added to the end-repaired dscDNA by DNA polymerase I, Large (Klenow) Fragment. A pair of Solexa adaptors was ligated to the repaired ends by T4 ligase and then 200-400 bp of fragments were selected using the Invitrogen 2% E-Gel. Specific dscDNA fragments were amplified by PCR and the libraries were then quantified and pooled.