alias: GSM3680573
title: GSM3680573: Breast cancer m6A-RIP IP biopsy 3; Homo sapiens; RIP-Seq
library_construction_protocol: (RNA purification) Total RNA was purified with the RNeasy kit from RNAi-depleted FTO (RNAi FTO) and control (RNAi Ctrl) cells. Residual DNA removal was performed using the RNase-Free DNase Set. (m6A-immunoprecipitation) Total RNA was then fragmented by mixing 90µl of RNA (at 1µg/µl) and 10µl of fragmentation buffer 10x (100mM Tris-HCl and 100mM ZnCl2) in thin-walled 200-μl PCR tubes, which were incubated at 94 °C for 35 seconds in a preheated thermocycler block with the heated lid opened. Then, 10 µl of 0.5M EDTA were added and the tubes were placed on ice. RNA was then subjected to sodium acetate precipitation and resuspended in 300μl of RNase-free water. RNA fragmentation size was assessed by running 0.5µg of RNA on a 2100 Bioanalyzer with an RNA Nano kit. Then, 2-5µg of fragmented RNA were kept at -80°C to later serve as input control. RNA was denatured by heat (70°C for 5 min, then on ice) and the immunoprecipitation mix was prepared as follows: 150µg of fragmented RNA in 755µl, 10µl (400 units) RNasin® Ribonuclease Inhibitors, 10µl 200mM Ribonucleoside Vanadyl Complex (RVC), 200µl IP buffer 5x (50mM Tris-HCl, 750 mM NaCl and 0.5% (vol/vol)), 7.8µl m6A antibody and 17.2µl RNase-free water. The mix was incubated overnight at 4°C on a rotation wheel. The next day, 60 µl of G-protein dynabeads were washed twice with IP buffer 1x supplemented with antiproteases and blocked by incubating the beads in wash buffer supplemented with 0.5 mg/ml BSA for 1h on a rotation wheel. The beads were washed again twice, then added to the immunoprecipitation mix and incubated for 2h at 4°C on a rotation wheel. The beads were washed three times with 1ml of wash buffer supplemented with 10µl of RNasin® and 10µl of RVC, then elution was performed by TriPure extraction and RNA was resuspended in 13µl of RNase-free water. Retro-transcription of 11µl of immunoprecipitated RNA was completed with the SuperScript II Reverse Transcriptase kit, according to manufacturer's instructions. Then, second strand synthesis was performed from cDNA with NEBNext mRNA Second Strand Synthesis Module, according to manufacturer's instructions. Finally, double-stranded DNA was purified using a MinElute PCR purification and quantified by Qubit. Sequencing library was prepared with MicroPlex Library Preparation Kit v2 x12 for both input and IP samples.
identifiers.namespace: GEO
identifiers.id: GSM3680573