GSM4824416: muscle_WGBS_rep2; Danio rerio; Bisulfite-Seq
For WGBS, 500ng of zebrafish gDNA and spiked in 0.025ng of unmethylated lambda phage DNA (Promega, Madison, WI, USA) was sonicated to an average insert size of 300bp. Sonicated DNA was end repaired followed by ligation of adapters to DNA overnight using NEXTFLEX Bisulfite-Seq Barcodes (PerkinElmer, Waltham, MA, USA). DNA was bisulfite converted using EZ DNA Methylation Gold Kit (Zymo Research,Irvine,CA,USA ) according to manufacturer instructions. Library amplification was performed with KAPA HiFi HotStart Uracil+ DNA polymerase (Kapa Biosystems, Woburn, MA), using 8 cycles of amplification. Library concentration was quantified through qPCR using KAPA Library Quantification Kit (Sigma-Aldrich, St. Louis, MO, USA) according to manufacturer instructions. The combined libraries with 15% PhiX spike-in was sequenced on the Illumina HiSeq 10X platform using 150bp paired-end sequencing (high output mode). embryos/tissues were snap frozen in liquid nitrogen and stored at -80°C prior to DNA extraction. Genomic DNA (gDNA) was extracted using the QIAGEN DNeasy Blood & Tissue Kit (QIAGEN, Chadstone, VIC, Australia) according to manufacturer instructions in two biological replicates.