GSM5351856: Dome_ctrl_H3K27ac_rep1; Danio rerio; ChIP-Seq
The ChIP procedure was based on a previously described method (Zhang et al., 2018a). Embryos were deyolked by repeatedly blowing with a 200 µl pipette and cell pellets were collected by spinning down at 5000 rpm for 5 min at 4℃. After centrifuge, cell pellet was lysed in 40 µl lysis buffer (0.5% NP-40, 0.5% Tween, 0.1% SDS and proteinase inhibitor) with pipetting up and down several times. 0.1 unit of MNase (Sigma, Cat N3755) was added for chromatin digestion at 37℃ for 5 min. The reaction was then terminated by adding 1 µl 0.5 M EGTA. IP sample was incubated with 1 µg H3K4me3 antibody (Homemade) (Zhang et al., 2016) and 2 µg H3K27me3 antibody (Active Motif, Cat 61017) overnight with rotation at 4℃. In the next day, the sample was incubated with 300 µg protein A or protein G dynabeads (Life technologies, Cat 10001D or 10003D) for 2hrs with rotation at 4℃. The beads were washed 5 times in 150 µl RIPA buffer and once in 150 µl LiCl buffer. After washing, spin tubes briefly and remove the supernatant. For each IP sample, resuspend beads with 27 µl ddH2O and 1 µl 10x Ex-Taq buffer (TaKaRa). 1 µl proteinase K (Roche, Cat 10910000) was then added and the mix was incubated at 55℃ for 90 min to elute DNA from beads. The supernatant was then transferred to a new tube and the proteinase K was inactivated at 72℃ for 40 min. 1 µl rSAP (NEB, Cat M0371) was then added to dephosphorylate 3' end of DNA at 37℃ for 1hr. rSAP was inactivated at 65℃ for 10 min. The resulting sample was subjected to TELP library preparation as previously described (Peng et al., 2015). 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.