GSM8528242: WT-1k-biol rep 2; Danio rerio; ATAC-seq
Chorions were removed by 15 - min. incubation in Pronase E. 3. 30 embryos were deyolked manually with an eyebrow needle in Danieaus medium, centrifuged (500 x g, 5 min, 4°C), resuspended in 100 µl ice-cold PBS and centrifuged again. The cells were resuspended by pipetting in 50 µl ice-cold lysis buffer: (ATAC-RSB (10 mM TrisHCl pH 7.4, 10 mM NaCl, 3 mM MgCl2), with 0.1% NP40, 0.1% Tween20, and 0.01% digitonin)) and incubated on ice for 5 minutes, then diluted with dilution buffer (1 ml, ATAC-RSB containing 0.1% Tween-20). Nuclei were pelleted at 500 x g, 10 min, 4°C, and resuspended in 16.5 µl PBST. Tagmentation reaction was assembled according to the manufacturer instructions for Illumina small Tn5 buffer and enzyme kit in 50µl volume and incubated in a thermomixer for 30 min, 37°C, 800 RPM. The samples were purified using Qiagen PCR MinElute Purification kit. The purified transposed mix was pre-amplified for 5 cycles using NEBNext 2x Master Mix (NEB) and Nextera adapters and put on ice. To determine the number of additional cycles, 5 µl of the pre-amplified mixture was used to run a 15 ml qPCR. The amplified libraries were purified using SPRI beads (Beckmann) according to the manufacturer instructions. Omni-ATAC-seq