GSM4824390: dnmt3aa-3ab_cKO_48hpf_RRBS_rep2; Danio rerio; Bisulfite-Seq
RRBS library construction was performed using the restriction enzyme BccI and SspI (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 10U BccI and 10U SspI (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.