GSM3359496: oocyte_H3K4me1_rep2; Danio rerio; ChIP-Seq
The ChIP procedure (STAR ChIP-seq) was based on a previously described method with modifications to adapt it for zebrafish embryos (Gilfillan et al., 2012; Zhang et al., 2016). For oocyte and embryo samples, yolk was firstly removed as described above. 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), 2 µg H3K27ac antibody (Active Motif, Cat # 39133), 2 µg H3K27me3 antibody (Active Motif, Cat # 61017), or 2 µg H3K36me3 antibody (Active Motif, Cat # 61021), 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 2 hrs 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 1 h. 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 resulting sample is ready for TELP library preparation (Peng et al., 2015).
GEO
GSM3359496