GSM6774544: IgG_U2OS_RIP; Homo sapiens; RIP-Seq
Nuclear fractionation of Control-sh and METTL3sh-2 cells was carried out by washing the trypsinized cells in ice-cold PBS and resuspending them in ice-cold RLN1 buffer (50 mM Tris pH 8.0, NaCl 500 mM, MgCl2 1.5 mM and 0.5% NP-40) and after 5 min incubation for on ice, the samples were spun at 300g for 2 min at 4°C. The supernatant containing cytoplasmic fraction was discarded and RNA from the nuclear pellet was isolated using TRIzol reagent (Invitrogen).  RNA fragmentation followed by m6A RIP was performed with 15 µg of RNA spiked in with 9 ng of Bacterial RNA as previously described by Zeng et al 2018. Sequencing libraries were prepared using SMARTer Stranded Total RNA-Seq Kit V2 (Pico Input Mammalian) from Takara and single end sequenced (1X 75 bp) on illumina NextSeq 550 platform.  Total RNA isolated from Control-sh and METTL3 KD cells, 20-25 ug was directly sequenced on the Oxford nanopore platform. In brief Total RNA quantification and quality control was performed on Fragment Analyzer 5300 system and showed RNA integrity number (RIN) of 10 for both samples. Poly(A) RNA Selection Kit V1.5 (Lexogen) was used to enrich for polyadenylated transcripts with minor changes to the protocol. All the processing volumes were doubled, and 4ug of Control-sh or METTL3-KD RNA was used as an input in a single reaction. Each sample was batch-processed in 5 separate reactions that were combined at the final elution step. Control-sh and METTL3-KD poly(A)-enriched RNA was analyzed on Fragment Analyzer showing remaining rRNA contamination to be 11% and 21% of mass for each sample respectively. Enriched poly(A)-RNA was then purified using 1x of RNAClean XP (Beckman Coulter) beads to concentrate the sample and remove very short RNA fragments and eluted into 11ul of water. Qubit™ RNA High Sensitivity kit (Invitrogen) was used to quantify the RNA before direct RNA sequencing. SQK-RNA002 (ONT) kit was used for library preparation and followed according to the manufacturer's protocol. Briefly, 400ng of poly(A)-enriched RNA was used as an input, and recommended reverse-transcription step was performed. Control-sh and METTL3-KD libraries were loaded on individual FLO-MIN106 (ONT) flowcells and sequenced on GridION Mk1 device (ONT) for 72 hours yielding 1.65 and 1.35 million long-reads respectively. direct-RNA Nanopore sequencing