GSM6806742: Day 1, BioCAP, replicate 1; Petromyzon marinus; OTHER
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. BioCAP was performed on genomic DNA in replicate and with input controls, using methods described in Long et al. Methods Mol Biol 2018. Briefly, genomic DNA was sonicated to approximately 200 base pairs and incubated with biotinylated zinc-finger CxxC protein domain from human KDM2B. NMIs were eluted in buffer containing 700mM-1M NaCl, 0.1% Triton X-100, 20 mM HEPES pH 7.9 and 12.5% v/v glycerol, then DNA was purified using the Wizard SV Gel and PCR Clean-Up System (Promega) according to manufacturer instructions. Library preparation was performed on BioCAP samples using TruSeq ChIP Sample Preparation (Illumina) according to manufacturer instructions. Libraries were amplified using 18 PCR cycles and quantified using KAPA qPCR Library Quantification Kit (KAPA Biosystems) according to manufacturer instructions. Libraries were sequenced on the Illumina NovaSeq 6000 (150 bp, paired-end), generating 34M-307M read pairs per sample. BioCAP-seq