GSM8086924: NA3; Homo sapiens; RNA-Seq
Total RNA from 1million human CD4+ T cells cultured under different arginine conditions (FULL, LOW, NA) for 7 days were extracted using TRIzol (Takara) following the manufacturer's procedure. The quantity and quality of RNA were assessed by using NanoDrop ND-1000 (NanoDrop, Wilmington, DE, USA) and Bioanalyzer 2100 (Agilent, CA, USA) with RIN number >7.0, and confirmed by electrophoresis with denaturing agarose gel. Approximately 2ug of total RNA was used to remove ribosomal RNA. Following purification, the ribominus RNA was fragmented and reverse-transcribed to create the cDNA by SuperScriptTM II Reverse Transcriptase (Invitrogen, cat. 1896649, USA), which were next used to synthesize U-labeled second-stranded DNAs with E. coli DNA polymerase I (NEB, cat.m0209, USA), RNase H (NEB, cat.m0297, USA) and dUTP Solution (Thermo Fisher, cat.R0133, USA). An A-base is then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contains a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. Single- or dual-index adapters are ligated to the fragments, and size selection was performed with AMPureXP beads. After the heat-labile UDG enzyme (NEB, cat.m0280, USA) treatment of the U-labeled second-stranded DNAs, the ligated products are amplified with PCR. The average insert size for the final cDNA library was 300±50 bp.