GSM2333028: N215S; Homo sapiens; RNA-Seq
RNA was isolated from purified CD45+CD3+ leukocytes by cell sorting using antibodies listed in Supplementary Table 6. After approximately two thousand cells were sorted into 96-well plates with 2.5 μl PBS (containing 0.5 μl RNaseOut (Life Technologies) and 0.5 μl dithiothreitol (DDT, Invitrogen), cytoplasm RNA was isolated as described previously (Pan, X., et al, Proc Natl Acad Sci USA, 2013). In brief, 2.5 μl of 2× selected cytoplasm lysis buffer (SCLB) was added and the cells were lysed by pipetting. Lysate solution was centrifuged at 8,000 rpm for 5 min at 4°C. Supernatant (~5 μl), containing the total cytoplasm RNA, was transferred to a PCR tube strip with individually attached dome caps. 5′-phosphorylated oligo-GdT24 (pGdT24) primer was used to selectively reverse transcribe mRNAs. The first strand cDNA was synthesized with Superscript Reverse Transcriptase III (Invitrogen) followed by second strand synthesis. The cDNA was purified with the Genomic DNA Clean & Concentrator kit (Zymo Research). Afterwards, several steps including DNA end-blunting, 5′-end phosphorylation and ligation were performed with the End-It DNA End-Repair Kit (Epicentre) with T4 DNA ligase (Epicentre). The product was directly amplified using REPLI-g UltraFast Mini Kit (QIAGEN) and purified using the same Genomic DNA Clean & Concentrator kit (Zymo Research Corp, Irvine, CA) column. Products were fragmented to 200-500 bp by a Bioruptor Sonicator (Diagenode). Fragment sizes were validated using the High Sensitivity DNA kit (Agilent Technologies) on an Agilent 2100 Bioanalyzer. Functional concentrations were determined by qPCR with standards for Illumina sequencing libraries. RNAseq libraries were generated by Center for Cancer Computational Biology (CCCB) at Dana-Farber Cancer Institute following manufacture protocol.