title: GSM6050583: HL-60(m7G MeRIP Transcriptome Seq), IP; Homo sapiens; OTHER
library_construction_protocol: Cells in logarithmic growth phase were collected, and we extracted total RNA using TRIzol reagent,The mRNA was isolated from total RNA with oligo(dT) magnetic beads (ThermoFisher), and then subjected to immunoprecipitation with the GenSeqTM m7G-IP Kit (GenSeq Inc., China) by following the manufacturer's instructions. RNA was decapping with Tobacco Decapping Enzyme, and then randomly fragmented to ~200 nt by RNA Fragmentation Reagents. Protein A/G beads were coupled to the m7G antibody by rotating at room temperature for 1 h. The RNA fragments were incubated with the bead-linked antibodies and rotated at 4°C for 4 h. The RNA/antibody complexes are then digested with Proteinase K and the eluted RNA is purified by phenol:chloroform extraction. RNA libraries for IP and input samples were then constructed with NEBNext® Ultra II Directional RNA Library Prep Kit (New England Biolabs, Inc., USA) by following the manufacturer's instructions. Libraries were qualified using Agilent 2100 bioanalyzer and then sequenced in a NovaSeq platform (Illumina).