GSM3402614: DAM-zGFP; Danio rerio; OTHER
We followed the protocol reported for medaka (Gutiérrez-Triana et al., 2016). WT zebrafish and medaka embryos were injected with each DAM-TF mRNA and its gDNA fraction was isolated when they reached the 80% epiboly stage. 40 embryos were used for each indipendent DamID replicate. gDNA was fragmented using DpnII restriction enzymes, dephosphorilated with phosphatase alkaline and cleaved at its GATC methylated sites using DpnI. Adapters, produced by hybridazing two oligos, were ligated to both ends for each fragment to build up DNA libraries through a 25-cycle PCR amplification step using a phosphorothioate primer. Each PCR product was run on a 1% agarose gel to check for the presence of a smear in DpnI-treated samples. Each library was amplified then five times before deep sequencing (Illumina HiSeq 2500 sequencing). Remaining DNA templates were removed with T7 exonuclease before sequencing. Three replicates for each sample were used for the iDamID-seq analysis. DNA libraries were prepared for sequencing using standard Illumina protocols.
GEO Accession
GSM3402614