Vahts mrna capture beads with oligo dt

Total RNA was isolated and purified using TRIzol (Life, cat.265709, CA, United States) following the manufacturer’s procedure. After the quality inspection of Agilent 2,100 Bioanalyzer (Agilent, cat. G2939AA, CA, United States) and NanoPhotometer^® (Implen, cat. N60, Munich, Germany), mRNA with poly(A) is purified from 1 μg total RNA using VAHTS^® mRNA Capture Beads with Oligo (dT) (Vazyme, cat. N401-01, Nanjing, China) through two rounds of purification.

Total RNA was isolated and purified using TRIzol (Life Services Inc., Glendale, CA, USA) following the manufacturer’s procedure. After the quality inspection of Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and NanoPhotometer^® (IMPLEN GmbH, Munich, Germany), poly (A) RNA was purified from 1μg total RNA using VAHTS^® mRNA Capture Beads with Oligo (dT) (Vazyme, Nanjing, China) using two rounds of purification. Then the poly(A) RNA was fragmented into small pieces using VAHTS^® Universal V6 RNA-seq Library Prep Kit (Vazyme, Nanjing, China) at 94°C for 8 min. Then the cleaved RNA fragments were reverse-transcribed to create the cDNA with reverse transcription reagent, which was next used to synthesize U-labeled second-stranded DNAs. An A-base was then added to the blunt ends of each strand, preparing them for ligation to the indexed adapters. Each adapter contained a T-base overhang for ligating the adapter to the A-tailed fragmented DNA. After the heat-labile UDG enzyme treatment of the U-labeled second-stranded DNAs, size selection was performed with VAHTS^® DNA Clean Beads (Vazyme, Nanjing, China). Then the ligated products were e amplified with PCR. Finally, we performed the 2×150bp paired-end sequencing (PE150) on an Illumina Novaseq™ 6000 (Illumina, Inc., San Diego, CA, USA) following the manufacturer’s recommended protocol.