GSM8257784: WT-2.Input; Mus musculus; RIP-Seq
Total RNA from ovaries and sorted oocytes was isolated using the NucleoZOL reagent (Macherey Nagel, 740404.200) and RNeasy Mini kit,according to the manufacturers' instructions.Polyadenylated RNA was isolated by two rounds of purification using the Dynabeads® mRNA Purification Kit (Thermo Fisher Scientific, 61006) according to the manufacturer's protocol. ac4C-RIP-seq was performed as described previously (Daniel Arango et al., 2018), with minor modifications. The library construction were standard Smart-seq2 protocol. Briefly, polyA RNA in cell lysis were first hybrided with Oligo-dT primer, then reverse transcripted by the PrimeScript Ⅱ RTase and TSO primer. The producted cDNA were pre-amplified with KAPA HiFi HotStart Ready Mix (2×) & TSO-PCR primer. Purified intact cDNA were then fragmented and amplified with barcodes adding by using TruePrep DNA Lybrary Prep Kit V2 for Illumina (cat.#TD502, Vazyme).