GSM5662235: C45_M; Rattus norvegicus; MeDIP-Seq
For each sample, 500 ng of DNA were diluted in a total volume of 60µL of EB buffer (Qiagen, Hilden, Germany). DNA was then transferred to a 96-well plate and sheared for 1 hour using the Covaris Focused-ultrasonicator. DNA was purified using Ampure XP in 20% polyethylene glycol (PEG) beads to obtain fragments sized from 200-500 basepairs (Beckman-Coulter, Brea, USA). Library preparation was performed on the Bravo Automated Liquid Handling Platform (Agilent, Santa Clara, USA) using the TruSeq DNA PCR-Free Sample Preparation Kit (Illumina, San Diego, USA). Following end-repair and A-tailing, adapters were ligated overnight at room temperature. PCR-free library preparation allowed for the conservation of methylated cytosines for subsequent methylated DNA immunoprecipitation. Finally, DNA was resuspended in 35µL of EB buffer (Qiagen, Hilden, Germany). DNA was quality controlled using Qubit Fluorometric Quantitation and the DNA 1000 Bioanalyzer 2100 kit (Agilent, Santa Clara, USA) to verify concentration and fragment size (250-550bp). methylated DNA immunoprecipitation (meDIP): For each sample, 400ng of the sequencing library were diluted in a total volume of 50µL of IP Buffer (10mM sodium phosphate buffer, pH7.0, 140mM NaCl, 0.05% triton). DNA was denatured by incubation at 95˚C for 10 minutes, followed by the addition of 48µL ice-cold IP buffer and incubation on ice for 10 minutes. 2µL of anti-5-methylcytosine antibody (Eurogenetec, Liège, Belgium), diluted to 1/50 in IP buffer (1µL of antibody per 1µg of DNA ratio), was added to each sample. Immunoprecipitation reactions were incubated for 16 hours at 4˚C with overhead rotation. Following two 5 minute washes with 150uL of 0.1% BSA/PBS, 50µL of Dynabeads Protein G were incubated with 5µL of secondary antibody (rabbit anti-mouse IgG; Jackson Immunoresearch, West Grove, USA) in 45uL ice-cold IP buffer for 15 minutes at room temperature with overhead rotation. Beads were washed twice with IP buffer to remove unbound secondary antibody and resuspended in 50µL IP buffer. The antibody-bound beads were added to the immunoprecipitation reactions and incubated for 2 hours at 4˚C with overhead rotation. Beads were then washed 6 times with 150µL of ice-cold IP buffer and resuspended in 98.97µL of Proteinase K digestion buffer (TE with 0.5% SDS). Following the addition of 1.25µL Proteinase K (20mg/mL; Qiagen, Hilden, Germany), samples were incubated in a thermomixer for 2 hours at 55˚C with a rotation speed of 1250rpm. The reaction was then allowed to cool at room temperature for 15 minutes. Supernatant was collected and bead cleanup was performed using equal volume SeraMag beads with 30% PEG. DNA was resuspended in 35µL of EB buffer (Qiagen, Hilden, Germany). Two parallel rounds of the following PCR amplification cycle conditions were performed on each sample in order to reduce PCR amplification bias. The reaction mixes were as follows: 15µL DNA, 27µL H2O, 12µL 5X HF buffer, 1.5µL DMSO, 1.0µL paired-end primer (Illumina), 0.5µL Phusion High-Fidelity DNA polymerase (New England Biolabs), 2µL indexing primer (Illumina – specific to each sample). The amplification cycle was as follows: 98˚C for 1 minute, 12X (98˚C for 15 seconds, 65˚C for 30 seconds, 72˚C for 30 seconds), 72˚C for 5 minutes. Reactions from the same sample were pooled and bead cleanup was performed using SeraMag beads in 20% PEG (102µL of beads per 120µL of reaction). DNA was resuspended in a final volume of 35µL of EB buffer. Indexed meDIP libraries were pooled as part of a larger set of 60 samples, which were combined into 5 pools of 12 samples each. Samples were distributed evenly by sex, prenatal treatment, and tissue type. Next-generation sequencing was performed on the pools by the Genome Sciences Centre in Vancouver, BC, Canada. Each sample pool was run on one HiSeq lanes, which produced approximately 600,000,000 paired-end reads of 125 bases per lane.