HiSeq X Ten paired end sequencing; RNA-seq and RRBS of developing zebrafish larval brain and WGBS of 4 week old WT and dnmt3aa/dnmt3ab CRISPR/CAS9 KO brain
RNA-seq and RRBS of developing zebrafish larval brain and WGBS of 4 week old WT and dnmt3aa/dnmt3ab CRISPR/CAS9 KO brain
Whole brains were dissected from zebrafish larvae and adults before being snap frozen in liquid nitrogen and stored at -80Â°C Genomic DNA (gDNA) was extracted from brains using the QIAGEN DNeasy Blood & Tissue Kit (QIAGEN, Chadstone, VIC, Australia) according to manufacturer instructions. For RNA extraction, half the lysate from the first step of the DNA extraction from the QIAGEN DNeasy Blood & Tissue Kit was added to TRIsure (Bioline) and purified following manufacturer instructions. All experiments in this study were performed in biological replicates. 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.