GSM9610752: Human T cells donor 5, stimulated anti CD3/28; Homo sapiens; RNA-Seq
Cells were harvested and lysed in TRIzol reagent (ThermoFisher). Total RNA was then purified using Direct-zol RNA purification kit (Zymo Research). Further RNA processing and sequencing were done by sequencing provider, Innomics. Sample QC was performed with Agilent 4150 bioanalyzer to identify samples with RIN>7. 200ng qualified RNA from each sample was processed for library preparation. In brief, mRNA enrichment was performed on total RNA using oligo(dT)-attached magnetic beads. The enriched mRNA with poly(A) tails was fragmented using a fragmentation buffer, followed by reverse transcription using random N6 primers to synthesize cDNA double strands. The synthesized double stranded DNA was then end-repaired and 5'-phosphorylated, with a protruding 'A' at the 3' end forming a blunt end, followed by ligation of a bubble-shaped adapter with a protruding 'T' at the 3' end. The ligation products were PCR amplified using specific primers. The PCR products were denatured to single strands, and then single-stranded circular DNA libraries were generated using a bridged primer. The constructed libraries were quality-checked and sequenced after passing the quality control. The library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular. The DNBs were load into the patterned nanoarray and pair end 150 bases reads were generated in the way of sequenced by synthesis.