RNA-Seq of HaCaT
Total RNA was extracted using Trizol reagent (ThermoFisher, 15596018) following the manufacturers procedure.The total RNA quantity and purity were analysis of Thermo Fisher Qubit3.0 (ThermoFisher, Q33216) and Agilent 5300 Fragment Analyzer (Agilent, CA, USA, M5311AA), high-quality RNA samples with RIN number > 7.0 were used to construct sequencing library. After total RNA was extracted, mRNA was purified from total RNA (2ug) using mRNA Caprure Beads2.0 (Yeasen Cat.12629ES, CHN) with two rounds of purification. Following purification, the mRNA was fragmented into short fragments using magnesium ions at 94C (Yeasen Cat.12340ES97, CHN). Then the cleaved RNA fragments were reverse-transcribed to synthesize first-Strand cDNA by Reverse Transcriptase, which were next used to synthesise secondstranded DNAs with E.coli DNA polymerase I, RNase H and dUTP Solution (Yeasen Cat.12340ES97, CHN). 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. Dual-index adapters were ligated to the fragments, and the ligated products were amplified with PCR by the following conditions: initial denaturation at 98 for 1 min; 14 cycles of denaturation at 98 for 10 sec; annealing at 60 for 30 sec; and extension at 72 for 30 sec; and then final extension at 72 for 5 min. The average insert size for the final cDNA librarys were 40050 bp. In PCR amplification, strand-specificity was achieved by utilizing the characteristic of high-fidelity DNA polymerase to only amplify cDNA strands without U-base. PCR amplification products were purified by Hieff NGS DNA Selection Beads(Yeasen Cat.12601ES75, CHN). At last, we performed the 2150bp paired-end sequencing (PE150) on an Illumina Novaseq X Plus (LC-Bio Technology CO., Ltd., Hangzhou, China) following the vendor's recommended protocol.