GSM9010195: WT2-1h; Mus musculus; RNA-Seq
Total RNA was extracted using RNAiso Plus (Takara) according to the manufacturer's instructions. Triplicate biological replicate RNA samples of each time point were sent to the Novel Bioinformatic Co., Ltd for sequencing and analysis. In brief, RNA quality was assessed using an Agilent 2200 Bioanalyzer, and samples with an RNA integrity number (RIN) > 7.0 were deemed acceptable for cDNA library construction. Total RNA was extracted from the samples by Trizol reagent (Invitrogen) separately. The RNA quality was checked by Agilent 2200 and kept at −80°C. The RNA with RIN (RNA integrity number) > 7.0 is acceptable for cDNA library construction. The cDNA libraries were constructed for each RNA sample using the VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (vazyme, Inc.) according to the manufacturer's instructions. Generally, the protocol consists of the following steps: Poly-A containing mRNA was purified from 1ug total RNA using oligo(dT) magnetic beads and fragmented into 200-600 bp using divalent cations at 85℃ for 6 min. The cleaved RNA fragments were used for first- and second-strand complementary DNA (cDNA) synthesis . dUTP mix was used for second-strand cDNA synthesis, which allows for the removal of the second strand. The cDNA fragments were end repaired, A-tailed and ligated with indexed adapters. The ligated cDNA products were purified and treated with uracil DNA glycosylase to remove the second-strand cDNA. Purified first-strand cDNA was enriched by PCR to create the cDNA libraries. The libraries were quality controlled with Agilent 2200 and sequenced by DNBSEQ-T7 on a 150 bp paired-end run.