title: GSM8238718: Cerebrum, P7, female, F1i, m6A-seq, Ip; Mus musculus; OTHER
library_name: GSM8238718
library_construction_protocol: Tissues were collected and separately homogenized in the presence of TRNzol Universal reagent (TianGen, #DP424).Total RNA was extracted in Trizol reagent following the manufacturer's protocol. Poly(A)+ RNA was then isolated using Dynabeads mRNA Purification Kit (Invitrogen, #61006). Synthesized 3′-adapter encoded with 6-nt barcode was pre-adenylated using 5′ DNA Adenylation Kit (NEB, E2610S). 200 ng RNA was fragmented to approximately 150~200 nt using RNA fragmentation reagents (Invitrogen, #AM8740). Fragmented RNA was purified and then repaired using T4 Polynucleotide Kinase (NEB, #M0201S). After cleanup, end-prepared RNA was ligated to adenylated 3′-adapter by T4 RNA Ligase 2, truncated KQ (NEB, #M0373S) incubated at 25 ℃ for 2 h, followed by 4 ℃ overnight. The 3′-adapters with 6-nt barcode and 5'-adapters with 8-nt unique molecular identifier (UMI) were synthesized by Sangon Biotech and Hippo Bio, respectively. To remove redundant adapter, 2 ml of Lambda Exonuclease (NEB, #M0262S) and 1 ml of 5′ Deadenylase (NEB, #M0331S) were added to each ligation mix. The reaction was incubated at 30 ℃ for 30 min, then at 37 ℃ for 30 min and finally 70 ℃ for 10 min to inactivate the enzymes. Subsequently, the samples were pooled to perform cleanup together and 10% of the sample pool was reserved as input sample. Multiplexed m6A immunoprecipitation (IP) was conducted 2 rounds. After immunoprecipitation, input and IP samples were respectively ligated to pre-heated 5′-adapter with T4 RNA Ligase 1 (NEB, #M0204S), and subjected to reverse transcription using HiScript III 1st Strand cDNA Synthesis Kit (Vazyme, #R312-01) and RT primer without cleanup. Library amplification was conducted with KAPA HiFi HotStart ReadyMix (KAPA Biosystems, #KK2601) with the universal primer and the indexed primer.