GSM7766577: Mouse_ID_50_CTRL-78W; Mus musculus; Bisulfite-Seq
Approximately 25 mg of frozen liver tissue was cut on dry ice and immediately placed into a 2 ml vial with ceramic homogenization beads (MP Biomedicals, Lysing Matrix D) containing 360 ul of Qiagen buffer ATL (Qiagen GmbH, Hilden, Germany). The tissue was allowed to equilibrate to room temperature, 20 µl of 40 mg/ml Proteinase K was added and homogenized with a Precellys 24 homogenizer (Bertin Technologies, Villeurbanne, France) at 5500 rpm for 20 s. The homogenized sample tubes were placed in a 56°C thermomixer at gentle agitation for 3 h. After proteinase treatment, the samples were treated with RNAse A, and DNA was isolated using Qiagen's DNeasy® kit following the manufacturer's instruction. The isolated DNA was checked for initial purity (A260/280 of ≥ 1.8, A260/230 ≥ 1.0) using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, Delaware). Yield was determined by Qubit broad range dsDNA assay kit and protocol (Life Technologies, Carlsbad, CA). 500 ng DNA was digested overnight with MspI (300 ng) or TaqαI (200 ng). Each sample underwent an additional 2 hr digestion before restriction enzyme deactivation. Each restriction enzyme digested DNA sample was pooled and then bead purified using Agencourt Ampure XP beads (Beckman Coulter Life Sciences, Brea, CA), and Qubit quantitation was used to determine the amount of λ for bisulfite methylation efficiency spike-in. End-repair and adapter ligation were performed with the KAPA Hyper Prep kit (Roche, Basle, Switzerland), along with a custom synthesized duplex adapter (Integrated DNA Technologies (IDT), Morrisville, NC). After overnight ligation and subsequent bead cleanup, the samples went through two rounds of bisulfite conversion and clean-up (Qiagen Epitect Bisulfite conversion kit, Germantown, MD). Library amplification was carried out using KAPA HIFI HotStart Uracil+ and IDT synthesized index and universal primer for 12 cycles, followed by bead cleanup. Library quality and yield were determined with the Agilent Bioanalyzer HS DNA kit (Santa Clara, CA), with the library size range predominately between 0 and 600bp. The libraries were sequenced on an Illumina NextSeq (San Diego, CA) by the National Health and Environmental Effect Research Laboratory (NHEERL) Genomics Research Core (GRC) at the US Environmental Protection Agency using the NextSeq 500/500 75 cycle kit.