medaka d-rR blastula DNase I-seq
Cells from 5000 d-rR strain medaka embryos were dissociated using a 21G needle and harvested by centrifugation at 500xg for 5 min. After washing with PBS, cells were resuspended in 500 μl of buffer A [15mM Tris-HCl (pH 8.0), 15mM NaCl, 60 mM KCl, 1mM EDTA, 0.5mM EGTA, 1mM PMSF]. Cells were isolated using a cell-strainer (Falcon, 352235), centrifuged at 500xg for 10 min, and resuspended in 1.5 ml of lysis buffer [buffer A with 0.1% IGEPAL CA-630]. After a 1 min incubation at 4℃ and nuclei were collected by centrifugation at 500xg for 10 min. Nuclei were washed in buffer A, resuspended in nuclear storage buffer [20 mM Tris-HCl pH 8.0, 75 mM NaCl, 0.5 mM EDTA, 50% (v/v) glycerol, 1 mM DTT, and 0.1mM PMSF] and stored at -80℃. For DNase I digestion, frozen nuclei were thawed on ice, washed in buffer A with 0.5mM spermidine and 0.3mM spermine, incubated for exactly 2 min at 37℃ in 3.5 ml of buffer D [1 volume of 10x DNase I digestion buffer with 9 volume of Buffer A] containing 480 U of DNase I. The reaction was stopped by adding stop buffer [50 mM Tris-HCl, pH 8.0, 100 mM NaCl, 0.1% SDS, 100 mM EDTA, 20 ug/ml RNase A, 0.5 mM spermidine, and 0.3 mM spermine] and proteinase K, and incubated at 55℃ overnight. Digested DNA was purified by phenol chloroform, sucrose fractionated, and fragments below 1 kb were pooled, end-repaired, ligated with adapters compatible with the Illumina sequencing platform and sequenced as single-end tags on HiSeq 1500 platform (Illumina).