title: GSM7883108: RNA-seq of chromatin associated RNAs with FP treatment, Rep1; Mus musculus; RIP-Seq
library_construction_protocol: Cells were washed with chilled PBS and pelleted by centrifugation before they were subjected to cell membrane lysis buffer (0.15% NP-40, 10 mM Tris-HCl pH 7.0, 150 mM NaCl, 25 μM α-amanitin, 10 U SUPERase_In) and incubated on ice for 5 min. Cell lysates were then added to 2.5 volumes of chilled sucrose cushion (10 mM Tris-HCl pH 7.0, 150 mM NaCl, 25% sucrose, 25 μM α-amanitin, 20 U SUPERase_In) and the mixture was centrifuged at 4 °C with 16,000 ×g for 10 min. The supernatant was the cytoplasmic fraction, and the pellet was regarded as cell nuclei. The nuclear pellet was then washed with nuclear wash buffer (0.1% Triton X-100, 1 mM EDTA, in 1× PBS, 25 μM α-amanitin, 10 U SUPERase In, and protease inhibitor mix) twice and pelleted by centrifugation at 1150 ×g for 2 min. The supernatant was discarded, and the pellet was subjected to chilled glycerol buffer (20 mM Tris-HCl pH 8.0, 75 mM NaCl, 0.5 mM EDTA, 50% glycerol, 0.85 mM DTT, 25 μM α-amanitin, 10 U SUPERase_In). Equal volumes of chilled nuclei lysis buffer (1% NP-40, 20 mM HEPES pH 7.5, 300 mM NaCl, 1 M urea, 0.2 mM EDTA, 1 mM DTT, 25 μM α-amanitin, 10 U SUPERase_In) were then added to the nuclei suspension and incubated on ice for 2 min followed by centrifugation at 4 °C at 18,500 ×g for 2 min. The pellet was subjected to chilled chromatin resuspension solution (25 µM α-amanitin, 20 units SUPERase_In, 1× protease inhibitor mix (in 1× PBS)) to obtain the insoluble chromatin-associated fraction and subjected to RNA extraction using TRIzol Reagent. For m6A immunoprecipitation, 50 μg chrRNA samples were fragmented by RNA fragmentation buffer (100 mM Tris-HCl pH 7.0 and 100 mM ZnCl2) at 94 °C for 30 s. Flowing standard ethanol precipitation and 1/20 of the fragmented RNA was saved as the input. The remaining fragmented RNA was immunoprecipitated with anti-m6A antibody (Abcam) in IP buffer (0.1% NP-40, 10 mM Tris-HCl pH 7.0, 150 mM NaCl) at 4 °C for 4 h. Dynabeads protein A/G (NEB) were washed, added to the mixture and incubated for 2 h at 4 °C with rotation. The m6A-containing RNA fragment was eluted twice with 6.7 mM N6-methyladenosine 5'-monophosphate sodium salt at 4 °C for 1 h and precipitated with ethanol at -80 °C overnight. RNA was dissolved in 10 μl nuclease-free water and subjected to reverse transcription for qPCR (see reverse transcription and quantitative Real Time-PCR). For chrRNA m6A-seq, cDNA library construction and sequencing were performed at LC-BIO Biotech Ltd. (Hangzhou, China). Paired-end 2 × 150 bp sequencing on an Illumina NovaSeq™ 6000 platform was also performed on libraries.