GSM4411562: Hmx3-Cre/tdTomato/Dnmt1loxp_WT_E16_MeDIP_1; Mus musculus; MeDIP-Seq
Mice were deeply anesthetized intraperitoneally with 10% chloral hydrate in phosphate-buffered saline (PBS; pH 7.4). Embryos of anesthetized timed pregnant dams were prepared as described in Zimmer et al. (2011). For FACS-mediated enrichment of tdTomato cells at E16, the whole basal telencephalon of living brains was subjected to cell dissociation. Briefly, embryonic brains were prepared in Krebs buffer (126mM NaCl, 2.5mM KCl, 1.2mM NaH2PO4, 1.2mM MgCl2, 2.1mM CaCl2, 10mM D-glucose, 12.5mM NaHCO3) and the basal telencephalon was dissected and collected in ice-cold Hanks balanced salt solution (HBSS; Invitrogen) supplemented with 0.65% glucose. After incubation with 0.025% trypsin (Invitrogen) in HBSS for 17 min at 37 °C, cells were dissociated by trituration and filtering through nylon gauze. Cells were washed with HBSS supplemented with 0.65% glucose prior to FACS. Following the addition of DAPI, cells were sorted using an ARIA III FACS sorter (BD Biosciences) with the maximal flow rate of 6. The tdTomato reporter was excited by a 561-nm yellow/green solid-state laser and emission signal was detected in a range of 579–593 nm. According to their FCS/SSC criteria followed by cell doublet exclusion via an FSC-H versus FSC-W criterium, DAPI-negative living cells were sorted based on a distinctive tdTomato signal. Cells of interest were collected in DMEM/F12, 10% FBS, 100 U/mL penicillin, 100 g/mL streptomycin at 4 °C and pelleted by centrifugation. Enriched tdTomato cells prepared from the E16 basal telencephalon were split for each brain to prepare RNA for RNA sequencing and DNA for methylation-specific DNA immunoprecipitation (MeDIP) sequencing in parallel from the same set of sample. For RNA isolation, pellets were dissolved in 500 μL Trizol Reagent (Life Technologies) and subsequentially frozen on dry ice. For MeDIP-Seq analysis, cell pellets were frozen at −80 °C until further use. For RNA isolation the samples were then subjected to standard RNA isolation procedure using Trizol Reagent (Life Technologies) with additional application of GlycoBlue (Thermo Fisher Scientific) to a final concentration of 0.2% during RNA precipitation for better visualization of the pellet, digested with RNase-free DNase (Qiagen) and checked for integrity by capillary gel electrophoresis (Bioanalyzer, Agilent Technologies, Inc.). DNA isolation of FACS-enriched tdTomato cells was performed using QIAamp DNA Micro Kit (Qiagen) according to manufacturer's instruction. For genome-wide methylation analysis, we applied immunoprecipitation methods for the enrichment of 5-methylcytosines. Specifically, 100 ng of genomic DNA was used as starting material. The Methylated-DNA IP Kit from Zymo (Cat. No. D5101) was applied according to manufacturer's instructions. The product of the IP and control reaction was then used for preparation of Illumina compatible libraries according to the TruSeq Nano DNA Library Prep Kit (Cat. No. FC-121-4001). Libraries were sequenced on a HiSeq 2000 yielding 50 bp single end reads. The sequencing reads were demultiplexed using the Illumina CASAVA tool and sequence quality was checked using FASTQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The reads were then aligned to the genome of Mus musculus (mm10) using Bowtie 2 (versions 2.0.2) with standard parameters. Differentially methylated regions were identified using the MEDIPS package for R (version 1.16.0; Lienhard et al. 2014) with a window size of 700 bp and a minimum coverage of 5% of the window length. A detailed description of the analysis pipeline can be found in Halder et al. (2016). Gene Ontology term enrichment analysis was performed for genes that were assigned to differentially methylated regions between wild-type and knockout samples. Results of gene ontology (GO) enrichment analysis were visualized in a bar diagram including the respective Benjamini–Hochberg corrected P value.
GEO Accession
GSM4411562