GSM4084050: OCM1.OE.HINT2.RNA-seq; Homo sapiens; RNA-Seq
For RNA-seq, total RNA was extracted from samples using an EZ-press RNA Purification Kit (B0004). For miCLIP-seq, mRNA was isolated from tumor and normal cell lines by using Dynabeads mRNA Purification Kit (Life Technologies, 61006). For RNA-seq, libraries were constructed from  100 ng of total RNA using an Illumina TruSeq RNA Sample Prep Kit (San Diego, CA, USA) following the manufacturer's protocol. For MeRIP-seq, purified mRNA was randomly fragmented to approximately 100 nucleotides using Ambion RNA fragmentation reagents and then subjected to IP with an anti-m6A antibody (202003, Synaptic Systems) and protein A magnetic beads (88845, Pierce) in meRIP buffer (150 mM NaCl, 10 mM Tris-HCl, pH 7.4, 0.1% NP-40) supplemented with RNase inhibitor. m6A-containing mRNA fragments were eluted with m6A in MeRIP buffer and purified using TRIzol reagent. For miCLIP-seq, mRNA was fragmented to ~100 nucleotides using fragmentation reagent (Life Technologies, AM8740), and incubated with 5 µg of an anti-m6A antibody (Abcam, ab151230) in 450 µL of IP buffer (50 mM Tris, 100 mM NaCl, 0.05% NP-40, adjusted to pH 7.4) under gentle rotation at 4°C for 2 h. The mixture was transferred to a clear flat-bottom 96-well plate (Corning) on ice and irradiated three times with 0.15 J/cm2 at 254 nm using a CL-1000 Ultraviolet Crosslinker (UVP). After irradiation, the mixture was collected and incubated with 50 µL of prewashed Dynabeads Protein A (Life Technologies, 1001D) at 4°C for 2 h. After extensive washing twice with high-salt buffer (50 mM Tris, 1 M NaCl, 1 mM EDTA, 1% NP-40, 0.1% SDS, adjusted to pH 7.4) and twice with IP buffer, the samples on the beads were subjected to dephosphorylation with T4 PNK (NEB, M0201L) at 37°C for 20 min. The RNA was then eluted from the beads by proteinase K (Sigma, P2308) treatment at 55°C for 1 h, followed by phenol-chloroform extraction and ethanol precipitation. The purified RNA was subjected to library construction using SMARTer smRNA-Seq Kit for Illumina (Clontech Laboratories, 635030) according to the manufacturer's instructions.