title: GSM4824384: uhrf1_cKO_48hpf_RRBS_rep2; Danio rerio; Bisulfite-Seq
library_construction_protocol: RRBS library construction was performed using the restriction enzyme MspI (New England BioLabs, Ipswich, MA, USA) and 0.025ng of unmethylated lambda phage DNA (Promega, Madison, WI, USA) was used as a spike-in control to determine the efficiency of bisufite conversion. Briefly, 500ng of zebrafish gDNA was digested for 2 hours with 20U MspI (New England BioLabs, Ipswich, MA, USA). The 5' overhangs of the digested DNA were filled in and A-tailed using klenow fragment exo-  (New England BioLabs, Ipswich, MA, USA) before 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 13 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.