GSM5741673: WGBS GABAergic neurons, MeCP2 KO, rep2; Mus musculus; Bisulfite-Seq
The brain tissues were rapidly dissected in ice-cold homogenization buffer and the INTACT procedure was applied to affinity purify GFP+ nuclei. For brain tissue of each mouse, half of the isolated nuclei were resuspended in 600 μl Lysis buffer (100 mM Tris-HCl, pH 8.5, 5 mM EDTA, 0.2% SDS, 200 mM NaCl) for DNA purification, 25 μl Proteinase K (Thermo Fisher, cat# EO0491) treatment at 55°C for overnight using an end-to-end rotator. The second day, the samples were incubated at 85°C for 45 minutes to inactive Proteinase K followed by the treatment of 5 μl RNase A for 1 hour at room temperature. The entire solution was then transferred to a pre-spun “Phase Lock Gel (PLG) 2 ml Heavy” tube. Equal volume of Phenol:Chloroform:Isoamyl Alcohol (25:24:1 Saturated with 10 mM Tris, pH 8.0, 1 mM EDTA) was added to samples, mixed completely, and centrifuged for 10 min at 12,000 rpm. The aqueous layer solution was transferred into a new Eppendorf tube and precipitated with 600 μL isopropanol. The pellet was washed with 75% ethanol, air-dried, and eluted with Nuclease-Free Water (Ambion). Another half of the beads-bonded nuclei were directly resuspended in TRIzol Reagent for RNA extraction following the standard protocol. For each cell type and genotype, two biological replicates were subjected to WGBS and RNA-Seq. The EZ DNA Methylation Gold kit was used for sodium bisulfite conversion prior to library preparation. All WGBS libraries were prepared from 100 ng nuclei DNA. Accel-NGS Methyl-Seq (Swift BioSciences) kit was used for library preparation according to the manufacturer's protocols. For each replicate, 4 μg of total RNA was used for RNA-seq library construction following instructions of Illumina mRNA sample prep kit. Briefly, total RNA was depleted of ribosomal RNAs, subjected to 5 min of heat fragmentation, and converted to strand-specific cDNA libraries using the TruSeq Total RNA library prep kit with RiboZero depletion (Illumina). Libraries were submitted for 150 paired-end sequencing on the Illumina HiSeq 2500 platform.