title: GSM2859234: WAT ENT m6A IP; Mus musculus; RNA-Seq
library_construction_protocol: RNA was isolated from mouse White Adipose Tissue using the TRIzol (Invitrogen) reagent by following the company manual. mRNA was isolated by using Dynabeads mRNA Purification Kit (Invitrogen) , fragmented and ethanol precipitated mRNA (5 μg) was incubated with 10 μg of anti-m6A polyclonal antibody (Synaptic Systems, 202003) in IPP buffer (150 mM NaCl, 0.1% NP-40, and 10 mM Tris-HCl [pH 7.4]) for 2 hr with rotatin at 4°C. The mixture was then immunoprecipitated by incubation with 50 μl protein-A beads (Sigma, P9424) at 4°C for an additional 2 hr. After being washed three times, bound RNA was eluted from the beads with 0.5 mg/ml N6-methyladenosine (BERRY & ASSOCIATES, PR3732) in IPP buffer and then extracted by Trizol. The remaining RNA was re-suspended in H2O and used for library generation with TruSeq™ RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer's protocol.The libraries were sequenced using HiSeq 2500 (Illumina) in paired-read mode, creating reads with a length of 101 bp. Sequencing chemistry v2 (Illumina) was used. Approximately 100 ng of recovered RNA was then used for library preparation using a TruSeq™ RNA Sample Prep Kit v2 (Illumina, San Diego, CA, USA) according to the manufacturer's protocol.The libraries were sequenced using HiSeq3000 (Illumina) in single-read or paired-read mode, creating reads with a length of 101 bp.
attributes.tag: GEO Accession
attributes.value: GSM2859234