title: GSM2805445: 532_M14WT_NPC_5% Input; Mus musculus; ChIP-Seq
library_construction_protocol: Positive ES clones were used for injection into c57 blastocysts and generation of chimerical mice. To produce Mettl14 f/+ mice, the chimeras were crossed wild-type c57 for germ line transmission and then crossed with Atcb-Flpe transgenic mice (The Jackson Laboratory, # 003800) to remove FRT flanked selection cassette. Male Mettl14 f/+ were crossed to female EIIa-Cre transgenic mice (The Jackson Laboratory, # 003724) to obtain Mettl14 +/- and Mettl14 +/- mice were intercrossed to obtain Mettl14 conventional knockout mice. To conditional knockout Mettl14 in brain, floxed Mettl14 mice were bred with nestin-Cre transgenic mice (The Jackson Laboratory, # 003771) to generate Mettl14 f/f;nes-cre. Mice were maintained at the SANFORD BURNHAM PREBYS Medical Discovery Institute Institutional animal facility, and experiments were performed in accordance with experimental protocols approved by local Institutional Animal Care and Use Committees (IACUC). Total RNA was isolated using TRIZOL (Invitrogen) and treated with DNaseI (Roche). Polyadenylated mRNA was purified using GenElute™ mRNA Miniprep Kit (Sigma-Aldrich) and residual ribosomal RNA was depleted with RiboMinus™ Eukaryote System v2 (Life Technologies). m6A protocol: Purified polyA+ RNA was blotted to a nylon membrane (Millipore) using Bio-Dot Microfiltration Apparatus (Bio-Rad). After crosslinking with a UV crosslinker (Spectroline), the membrane was blocked with non-fat milk in TBST and then incubated with antibody against m6A (Synaptic Systems, Cat. # 202 003, 1:1,000) then an HRP-conjugated antibody against rabbit IgG. After incubation with the Immobilon Western Chemiluminescent HRP Substrate (Millipore), the membrane was exposed to autoradiography film (Kodak). ChIP-seq: Histones were extracted from NSCs cultured for 7 days in vitro using Histone Extraction Kit (Abcam) following manufacturer’s protocols. Histone lysates were separated on SDS-PAGE gel, blotted onto Immobilon-FL PVDF membrane (Millipore, Cat. # IPFL00010) and incubated with primary antibodies including rat anti-H3 (Active Motif, Cat. # 61647; 1:2000), rabbit anti- H3K4me3 (Abcam, Cat. # Ab8580; 1:2000), rabbit anti-H3K27ac (Active Motif, Cat. # Ab8580; 1:2000), rabbit anti-H3K27me3 (Millipore, Cat. # 07-449; 1:2000), rabbit anti-H3K9me3 (Active Motif, Cat. # Ab8898; 1:2000) and rabbit anti-H3ac (Active Motif, Cat. # 39133; 1:2000). Secondary antibodies were conjugates of IRDye 680RD and 800CW (LI-COR Biosciences). Membranes were scanned by Odyssey Imager (LI-COR Biosciences) and quantified using Image Studio Lite (LI-COR Biosciences).
attributes.tag: GEO Accession
attributes.value: GSM2805445