alias: GSM3377770
title: GSM3377770: KO_0_3_MeRIP-seq input; Mus musculus; RIP-Seq
library_construction_protocol: RNA-Seq: RNA was harvested using Trizol reagent MeRIP-seq: Fragmented RNA was incubated for 2 hr at 4C with 0.5 mg/ml anti-m6A polyclonal antibody in IP buffer (150mM NaCl, 0.1% Igepal CA-630, 10 mM Tris-HCl [pH 7.4]) supplemented with RNasin Plus RNase inhibitor RNA-Seq: Sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample MeRIP-seq: First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-) . Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 150~200 bp in length, the library fragments were purified with AMPure XP system . Then 3 µl USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95 °C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) and library quality was assessed on the Agilent Bioanalyzer 2100 system.
identifiers.namespace: GEO
identifiers.id: GSM3377770