GSM5574727: normal rep13; 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. After purification by VAHTS DNA Clean Beads (Vazyme, cat.N411-02, Nanjing, China), quality control of concentration and fragment size is performed by Agilent 2100 Bioanalyzer (Agilent, cat.G2939AA, CA, USA) and Qubit assay tubes (Life, cat. 1604220, CA, USA).