title: GSM6094429: LOW_INPUT_INIT10; Homo sapiens; OTHER
library_construction_protocol: Total RNA was isolated from cell lines, peripheral blood or Murine heart tissue with TRIzol (Invitrogen) according to the manufacture's protocol. The mRNA was isolated by subjecting total RNA to oligo(dT) enrichment using Dynabeads Oligo(dT)25 (NEB) Fragmented mRNAs from different samples were first treated with T4 PNK. Then the samples were ligated with pre-adenylated 3'-linkerLs (5'-NNNNNNNNNNNNAGATCGGAAGAGCGTCGTG-3', Bold N represnts for designed 6-nt barcode sequences, Italic N represents for 6-nt random sequences). Extra 3'-linkerLs were removed by 5′-deadenylase and RecJf digestion. The reaction mixtures were subsequently pooled together and purified. 1/50 of the purified sample was retained as input control while the remaining sample was applied to m6A antibody immunoprecipitation (IP). The input control sample and the IP sample were reverse transcripted using RT primer (5'-ACACGACGCTCTTCCGATCT-3') to obtain cDNA samples. The cDNA samples were then ligated with 5'-adaptor (5'-Phos-NNNNNNNNNNAGATCGGAAGAGCACACGTCTG-SpC3-3'),followed by determination of PCR cycle number by RT-qPCR. Finally, library PCR amplification was performed using the NEB primers and the products were purified by magnetic beads or agarose gel electrophoresis.