GSM5284842: NK cells_m6A_Input_WT_rep3; Mus musculus; RIP-Seq
For m6A-seq, total RNA was isolated by TRIzol reagent (Thermo Fisher Scientific) from fifty million IL-2 expanded NK cells. Polyadenylated RNA was further enriched from total RNA by using Dynabeads® mRNA Purification Kit (Invitrogen). mRNA samples were fragmented into ~100-nucleotide-long fragments with RNA fragmentation reagents (Invitrogen). Fragmented mRNA (5 μg mRNA) was used for m6A immunoprecipitation (m6A-IP) using an m6A antibody (Synaptic, 202003) following the standard protocol of the Magna MeRIP m6A Kit (Merck Millipore). For Ythdf2 RIP-seq, fifty million IL-2-expanded NK cells were harvested and washed twice with cold PBS, and the cell pellet was lysed with two volumes of lysis buffer [10 mM HEPES pH 7.6, 150 mM KCl, 2 mM EDTA, 0.5% NP-40, 0.5 mM DTT, 1:100 Protease Inhibitor cocktail (Thermo Fisher Scientific), and 400 U/mL SUPERase-In RNase Inhibitor (Thermo Fisher Scientific)]. The lysate was incubated on ice for 5 min and centrifuged for 15 min to clear the lysate. One-tenth volume of cell lysate was saved as input. The rest of the cell lysate was incubated with 5 μg anti-YTHDF2 (MBL, Cat No. RN123PW) that was pre-coupled with Protein A magnetic beads (Invitrogen, Cat: 10001D) at 4℃ for 2h with gentle rotation. Afterward, the beads were washed five times with 1 ml ice-cold washing buffer (50 mM HEPES pH 7.6, 200 mM NaCl, 2 mM EDTA, 0.05% NP-40, 0.5 mM DTT, and 200 U/mL RNase inhibitor). Immunoprecipitated samples were subjected to Proteinase K digestion in wash buffer supplemented with 1% SDS and 2 mg/mL Proteinase K (Thermo Fisher Scientific) incubated with shaking at 1200 rpm at 55℃ for 1 h. Total RNA was extracted from both input and immunoprecipitated RNA by adding 5 volumes of TRIzol reagent, followed by Direct-zol RNA Miniprep (Zymo Research). For m6A-seq,RNA was enriched through RNA Clean & Concentration- 5 (Zymo Research) for library generation with a KAPA RNA HyperPrep Kit (Roche). Sequencing was performed at the City of Hope Genomics Facility on an Illumina HiSeq2500 machine with single read 50 bp mode. For Ythdf2 RIP-seq, cDNA library generation was produced with a KAPA RNA HyperPrep Kit (Roche) and sequenced on Illumina HiSeq2500 platform.