title: GSM4411558: Hmx3-Cre/tdTomato/Dnmt1loxp_WT_E16_rna_1; Mus musculus; RNA-Seq
library_construction_protocol: 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. RNA quality was further assessed by measuring the RIN (RNA Integrity Number) using the fragment analyzer from Advanced Analytical (U.S.A.). Library preparation for RNA-Seq was performed using the TruSeq™ RNA Sample Prep Kit v2 (Illumina, Cat. N°RS-122-2002, U.S.A.) starting from 50 ng of total RNA. Accurate quantitation of cDNA libraries was performed by using the QuantiFluor™ dsDNA System (Promega, U.S.A.). The size range of final cDNA libraries was determined applying the DNA chip on the fragment analyzer (average 350 bp; Advanced Analytical). cDNA libraries were amplified and sequenced by using the cBot and HiSeq2000 from Illumina (SR; 1x50 bp; ~30-40 million reads per sample). Sequence images were transformed with Illumina software BaseCaller to bcl files, which were demultiplexed to fastq files with CASAVA v1.8.2. Quality check was done via fastqc (v. 0.10.0, Babraham Bioinformatics, G.B.). Read alignment was performed using STAR (v2.3.0; Dobin A et al. 2013) to the mm10 reference genome. Data were converted and sorted by samtools 0.1.19 and reads per gene were counted via htseq version 0.5.4.p3. Data analysis was performed using R/Bioconductor (3.0.2/2.12; Gentleman RC et al. 2004) loading DESeq2 (Love MI et al. 2014). Candidate genes were filtered to a FDR-corrected pvalue<0.05.
attributes.tag: GEO Accession
attributes.value: GSM4411558