GSM6165664: A2; Homo sapiens; RNA-Seq
Briefly, the concentration of total RNA was measured by Qubit RNA HS assay kit (Invitrogen, Q32852). Total RNA was fragmented into 100-200 nt RNA fragments using ZnCl2 Add DNase I to the RNA sample, digest at 37 °C for 30 min to remove the residual DNA in the sample, and purify the magnetic beads to recover the RNA. The 5XRT Buffer was added to sample RNA, ribosomal RNA probe was added, and the temperature gradient reaction fragments the RNA sample and remove rRNA. EpiScriptTM IV, RNase inhibitor, DTT, TSO, and random primers were added to the RNA samples in step2. Add 2XpfuMax HiFi PCR ProMix and sequencing primers to the first strand of cDNA sample, and then amplified in PCR apparatus after mixing. The PCR amplification product was purified in a 1X ratio using EpiTM DNA Clean Beads, and the purification product was recovered from 300-400 bp of DNA fragments in a 0.65X/0.2X ratio with magnetic beads. A second cycle of PCR amplification of the purified product was performed to enrich DNA fragments of 300-400 bp. Bioptic Qsep100 Analyzer was used to conduct quality inspection on the library to detect whether the size distribution of the library conforms to the theoretical size.