GSM6806756: Brain, WGBS, replicate 1; Petromyzon marinus; Bisulfite-Seq
For DNA extraction, lamprey embryos were lysed in buffer containing 20 mM Tris, pH 8.0, 100 mM NaCl, 15 mM EDTA, 1% SDS and 0.5 mg/ml proteinase K for 3 h at 55 °C. Lysis was followed by two phenol:chloroform:isoamyl alcohol (25:24:1) extractions and subsequent centrifugation (5 min at 17,949g). DNA was then precipitated by adding 0.2 volumes of 4 M ammonium acetate and 3 volumes of 96% ethanol. The reaction was left on ice for a minimum of 30 min. The DNA precipitate was centrifuged for 20 min at 4 °C (17,949g), and the pellet was washed with 500 μl of 70% ethanol and centrifuged for 5 min (17,949g) at room temperature. The pellet was then resuspended in 200 μl of TE buffer, and 1 μl of RNase A (20 μg/μl) was added. The reaction was left to proceed for 30 min at room temperature, after which time the DNA was precipitated with 0.1 volumes of 4 M ammonium acetate and 1 volume of isopropanol on ice for 2 h. The reaction was then centrifuged for 30 min at 4 °C (17,949g), and the pellet was resuspended in TE buffer. WGBS was performed on genomic DNA extracted from egg, sperm, day 1, day 2, brain, muscle and PBMC in replicate using methods described in Skvortsova et al. Nat Commun 2019. Briefly, DNA was spiked with unmethylated lambda phage fragments (Promega) and sonicated to approximately 300 base pairs. Sonicated DNA was concentrated in a vacuum centrifuge concentrator to the final volume of 20µL, required for bisulfite conversion with the EZ DNA Methylation-Gold Kit (Zymo Research). Bisulfite-converted DNA was then subjected to low input library preparation using the Accel-NGS Methyl-seq DNA Kit (Swift Biosciences). Briefly, the single-stranded, bisulfite-converted DNA was subjected to an adaptase reaction, followed by primer extension, adapter ligation (Methyl-seq Set A Indexing Kit-Swift Biosciences), and indexing PCR. Libraries were quantified using KAPA qPCR Library Quantification Kit (KAPA Biosystems) according to manufacturer instructions. Methylome libraries were sequenced on the Illumina HiSeqX platform (high-throughput mode, 150_bp, PE).