title: GSM8585165: m_KO_Th9_2; Mus musculus; RNA-Seq
library_construction_protocol: For m6A-seq, total RNA was isolated by TRIzol reagent. 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 Th9 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 bulk RNA-seq, total RNA was isolated by TRIzol reagent. 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). 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 NovaSeq 6000  machine with 100-bp paired-end reads (PE100). m6a-seq, RIP-seq, poly-RNA-seq
identifiers.namespace: GEO
identifiers.id: GSM8585165_r1