title: GSM2401620: WT-R1_RNA; Mus musculus; RNA-Seq
library_construction_protocol: ATAC-seq: Protocol as described in Buenrostro et al (2015): doi:10.1002/0471142727.mb2129s109, with the excpetion that the Nuclei EZ Prep Nuclei Isolation Kit (Sigma, NUC-101) was used for nuclei isolation. ATAC-seq: Protocol as described in Buenrostro et al (2015): doi:10.1002/0471142727.mb2129s109, using illumina barcoded primers for sequencing. RNA-seq: The cerebella of post natal day 7 Chd7f/f (cn) and Math1cre;Chd7f/f (cko) pups were dissected in DPBS (Gibco) and lobules IX and X were removed. The cerebella of the individuals from each genotype were pooled. Cells were dissociated using Papain I (100U, Worthington) followed by trituration. Granule cell precursors were isolated by Percoll fractionation with a 35% and 65% Percoll gradient (Sigma) as described (Yu T, Meiners LC, Danielsen K, Wong MT, Bowler T, Reinberg D, Scambler PJ, van Ravenswaaij-Arts CM, and Basson MA. Deregulated FGF and homeotic gene expression underlies cerebellar vermis hypoplasia in CHARGE syndrome. eLife. 2013;2(e01305.). Total RNA was extracted from freshly isolated cells (5x106/cerebellum) with an Agilent Absolutely RNA miniprep kit (Agilent Technologies, Stockport, UK) RNA-seq: RNA quality and integrity was assessed on a 2100 Bioanalyzer and 2200 Tapestation using an R6K screenTape (Agilent Technologies, Stockport, UK). After quantification on Qubit using Quant-iT RNA assay kit (Life Technologies, Paisley, UK), 2g total RNA was used from each sample for library preparation using an Illumina TruSeq kit (Illumina, Cambridge, UK). Libraries were resolved by agarose gel electrophoresis, 250-550bp DNA fragments isolated and gel purified with a Qiagen DNA gel extraction kit (Qiagen, Manchester, UK). Two libraries with unique barcodes were combined and sequenced per lane on a HiSeq2000 (Illumina, Cambridge, UK) at the Biomedical Research Centre, King’s Health Partners. ChIP-seq: The cerebella of post natal day 7 Chd7f/f (cn) and Math1cre;Chd7f/f (cko) pups were dissected in DPBS (Gibco) and lobules IX and X were removed. The cerebella of the individuals from each genotype were pooled. Cells were dissociated using Papain I (100U, Worthington) followed by trituration. Granule cell precursors were isolated by Percoll fractionation with a 35% and 65% Percoll gradient (Sigma) as described (Yu T, Meiners LC, Danielsen K, Wong MT, Bowler T, Reinberg D, Scambler PJ, van Ravenswaaij-Arts CM, and Basson MA. Deregulated FGF and homeotic gene expression underlies cerebellar vermis hypoplasia in CHARGE syndrome. eLife. 2013;2(e01305.). ChIP-seq: Freshly isolated cells (2-3x107) of each genotype were fixed, chromatin isolated, precipitated with an antibody to H3K4me1 (Abcam, Cambridge UK) and ChIP-seq libraries produced and sequenced as previously described (Tee WW, Shen SS, Oksuz O, Narendra V, and Reinberg D. Erk1/2 activity promotes chromatin features and RNAPII phosphorylation at developmental promoters in mouse ESCs. Cell. 2014;156(4):678-90.).
identifiers.namespace: GEO
identifiers.id: GSM2401620