GSM8149659: RNA_bisulfite-seq_BrainGF3; Mus musculus; OTHER
Total RNA was extracted using Trizol reagent (#AG21102, Accurate Bioeng. Co., Ltd., Hunan, China) and the mRNA was purified using the Dynabeads® mRNA Purification Kit Dynabeads Oligo(dT)25 (#61002, Invitrogen). mRNA was treated by DNaseⅠ buffer (AM8170G, Thermo Fisher Scientific) and Turbo DNase (Thermo Fisher Scientific, AM2238) at 37 ℃ for 10 min and purified by ethanol precipitation to remove genomic DNA. 1 μg of poly(A) RNA was fragmented into ~200 nt nucleotide fragments. According to the EZ RNA Methylation Kit protocol (#R5001, ZYMO RESEARCH), the poly(A) RNA fragments underwent sodium bisulfite treatment, desalting, and desulfonation reactions, leading to the construction of a strand-specific Bisulfit-seq library. The bisulfite-converted library was initiated with random hexamers (ACT) to reverse transcribe RNA samples. The first strand cDNA of the bisulfite-converted library was synthesized using 5× first-strand buffer (#18080044, Invitrogen), 10 mM DTT (#18080044, Invitrogen), 10 mM dNTP mix (#N0447L, NEB), 40 U/μl RNase Inhibitor (#M0307L, NEB), and reverse transcriptase III (#18080044, Invitrogen). The second strand cDNA synthesis was performed using the VAHTS Universal V6 RNA-seq Library Prep Kit for Illumina (#NR604-02, VAHTS), and the necessary adapters for library preparation were provided by the VAHTS® RNA Adapters set3 for Illumina.