GSM5351840: Shield_ctrl_H2AK119ub_rep1; Danio rerio; OTHER
CUT&RUN was conducted based on previously published protocol (Xia et al., 2019) with modifications in cell permeation to adapt it for zebrafish embryos. Embryos were deyolked by tweezers manually and transferred into 1.5 mL conventional, non-low-binding tube (Axygen). Then tubes were flicked several times to disperse cells in embryos, and resuspended by 60 ul washing buffer (20 mM HEPES-KOH, pH = 7.5; 150 mM NaCl; 0.5 mM Spermidine and Roche complete protease inhibitor). 10 µl Concanavalin-coated magnetic beads (Polyscience, Cat 86057) for each sample were gently washed twice, resuspended by binding buffer (20 mM HEPES-KOH, pH = 7.5; 10 mM KCl; 1 mM CaCl2; 1 mM MnCl2), and added carefully to the cells. The cells with beads were incubated at 23℃ for 30 min on Thermomixer (Eppendorf) at 400 rpm, then held at magnetic stand to exclude buffer, and resuspended by 75 µl antibody buffer (washing buffer supplied with 0.02% digitonin and 2 mM EDTA, freshly made) with H3K4me3 (validated homemade) (Zhang et al., 2016) antibody, or H3K27me3 antibody (Active Motif, Cat 61017), or H2AK119ub antibody (CST, Cat 8240s) diluted at ratio of 1:100. Then the samples were incubated at 4℃ on Thermomixer overnight at 400 rpm. On the second day, the samples were washed by digitonin-washing buffer several times on magnetic stand, and resuspended with 50 µl digitonin-washing buffer supplied with 700 ng/ml pA-MNase, which is kindly provided by Dr. Steven Henikoff, and incubated at 4℃ on Thermomixer for 3hrs at 400 rpm. After that, the cells were washed by digitonin-washing buffer on magnetic stand, and resuspended by 100 µl digitonin-washing buffer on ice for at least 2 min. Targeted region digestion was activated by adding 2 µl 100 mM CaCl2 for 30 min in ice, then stopped by 100 µl 2 X stop buffer (340 mM NaCl; 20 mM EDTA, pH = 8.0; 4 mM EGTA, pH = 8.0; 50 µg/ml RNase; 100 µg/ml glycogen; 0.02% digitonin suppled with spike-in DNA) and fully vortexed. To release fragments, the samples were incubated at 37℃ on Thermomixer at 400 rpm for 20 min. Then the supernatants were purified by phenol chloroform and ethanol purification, and subjected to Tru-seq library construction using NEBNext Ultra II DNA Library Prep kit (NEB, Cat E7645S) as standard protocols. The amplified DNA was size-selected using AMPure Beads for 200-800 bp DNA fragments. All libraries were sequenced by Illumina Hi-Seq 1500 or 2500 or XTen platform according to manufacturer's instruction.