GSM5768763: CAFS3; Homo sapiens; RNA-Seq
Total RNA was isolated and purified using TRIzol(Life, cat.265709, CA, USA) following the manufacturer's procedure. After the quality inspection of Agilent 2100 Bioanalyzer (Agilent, cat.G2939AA, CA, USA) and NanoPhotometer ® (Implen, cat.N60, Munich, Germany), mRNA with poly(A) is purified from 1μg total RNA using VAHTS ® mRNA Capture Beads with Oligo (dT) (Vazyme, cat.N401-01, Nanjing, China) through two rounds of purification. Subsequently, mRNA fragment was interrupted using VAHTS ® Universal V6 RNA-seq Library Prep Kit (Vazyme, cat.NR604, Nanjing, China) under 94℃ 8min and reversed transcription into cDNA which would use to synthesise U-labeled second-stranded DNAs. An A-base was added to the blunt ends of each strand to ligase the indexed adapters which contains a T-base at the tail end. After UDG enzyme treatment of the U-labeled double-strand DNA, size selection was performed with VAHTS ® DNA Clean Beads (Vazyme, cat.N411, Nanjing, China). Then the ligated products are amplified with PCR by the following conditions: initial denaturation at 98℃ for 5 min; 12-17 cycles of denaturation at 98℃ for 10 sec, annealing at 60℃ for 30 sec, and extension at 72℃ for 30 sec; final extension at 72℃ for 5 min. The average insert size of cDNA library was 280±80 bp.