GSM2523041: TAB-seq_ventral_DG_standard_housing_rep2; Mus musculus; OTHER
Tissue collection consisted of rapid removal of the brain, followed by flash freezing and storage at -80C. Frozen brains were sliced coronally at 200 µm thickness until reaching bregma -2.30. The brains were then removed from mounting position, rotated, and remounted to the mounting position for horizontal slicing ventral dentate tissue. Horizontal sections were sliced from interaural 3.24 mm to 0.92 mm26. A 300 µm diameter puncher was used to punch dorsal and ventral dentate gyrus region separately. RNA and DNA extraction were performed from the same sample using Qiagen Allprep DNA/RNA Mini kit (Qiagen, Cat# 80204.).  We performed on-column DNase I treatment during RNA extraction and on-column RNaseA treatment during DNA extraction. RNA was examined by Bioanalyzer 2100 (Agilent technologies, Santa clara, USA). TAB-seq: DNA from the same samples was separated for TAB-seq and MethylC-seq library preparation. Protection and oxidation portions of library preparation were performed in-house using the Wisegene kit as described in Yu et al6. Three spike-in control DNAs, lambda DNA (Cat# D1501, Promega), 5mC (Cat# S001, Wisegene) and 5hmC (Cat# S002, Wisegene) were added to each sample (2.5 µg of total DNA) before DNA shearing, in order to evaluate the bisulfite conversion efficiency, the protection rate of 5 hmC, and the oxidation rate of TET. Bisulfite conversion was then performed at the Genome Quebec Innovation Centre on the processed TAB-seq sample, as well as 1 µg of DNA for the MethylC-seq library. Methylated and unmethylated DNA sets (Cat# D5017, pUC19 DNA set, Zymo research) were added as spike-in controls (2 ng spike-in control in 1 ug DNA) to evaluate bisulfite conversion efficiency. The whole genome bisulfite sequencing (WGBS) libraries were prepared using NimbleGen SeqCap Epi Enrichment System (Cat# 07145519001, Roche NimbleGen, Inc.). Library amplification was done using KAPA HiFi Hotstart Uracil + DNA polymerase (Cat# KK2802, Kapa Biosystems).