GSM8863138: Cct7 KD Ctr 2; Mus musculus; RNA-Seq
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.