title: GSM9087660: HepG2 cells as controls [C1.IP]; Homo sapiens; OTHER
library_construction_protocol: In summary, 5 µg of fragmented mRNAs were retained as an input control for RNA sequencing, while 500 µg of fragmented mRNAs were incubated with 5 µg of anti-m6A polyclonal antibody (Synaptic Systems, 202003) in IPP buffer (comprising 150 mM NaCl, 0.1% NP-40, 10 mM Tris–HCl, pH 7.4) for 2 hours at 4°C. Subsequently, the mixture underwent immunoprecipitation through incubation with protein-A beads (Thermo Fisher) at 4°C for an additional 2 hours. The mRNAs bound to the beads were eluted using N6-methyladenosine (BERRY & ASSOCIATES, PR3732) in IPP buffer and extracted with Trizol reagent (Thermo Fisher) according to the manufacturer's instructions. The purified mRNAs were employed for RNA sequencing library preparation using the NEBNext® Ultra™ RNA Library Prep Kit (NEB). Both the input sample (without immunoprecipitation) and the m6A immunoprecipitated sample underwent 150 bp paired-end sequencing on an Illumina HiSeq sequencer.
identifiers.namespace: GEO
identifiers.id: GSM9087660_r1