GSM6284554: ShMETTL3-input MeRIP; Homo sapiens; RIP-Seq
The total RNA was extracted by an EASYspin Plus kit (Yishan, China) and dissolved in 40 µL of RNase-free water. Then, the cells were divided into two groups, one group for input and the other group was increased to 1 mL by the addition of buffer containing an RNase inhibitor, ribonucleoside vanadyl complexes, an m6A-specific antibody (Sigma–Aldrich) or rabbit IgG (Sigma Aldrich) and incubated for 2 h at 4 °C. Next, the prewashed beads A were added and incubated for 2 h. Elution buffer containing an anti-m6A antibody (Synaptic Systems) was added to the mixture and incubated for 1 hour with continuous shaking at 4 °C. The methylated mRNA was precipitated with 5 mg of glycogen and one-tenth volumes of 3 M sodium acetate in a 2.5 volume of 100% ethanol at -80 °C overnight. For each sample, 1 μg of the total RNA was used for the RNA-seq library preparation by a VAHTS Stranded mRNA-sequence Library Prep Kit (Vazyme, China). Polyadenylated mRNAs were purified, fragmented and then converted into double-stranded cDNA. After end repair and A tailing, the DNA was ligated to VAHTS RNA Adapters (Vazyme, China). Purified ligation products corresponding to 200-500 bps were digested with heat-labile UDG, and the single strand cDNA was amplified, purified, quantified and stored at -80 °C before sequencing