GSM4431945: HiC_St5_mofRNAi_rep1; Drosophila melanogaster; Hi-C
Hi-C: D. melanogaster dechorionated embryos were fixed using 1.8 % formaldehyde for 15 min at RT. 50-100 embryos were hand staged based on morphology for the late embryonic stage 15 (14-16h AEL) or the mid-nuclear cycle 14 stage. The latter stage contains nuclei purely to S phase. The next mitosis takes place during gastrulation (Yuan et al., 2016). We employed a modified version of the in situ Hi-C protocol (Rao et al., 2014) described in Ramírez et al. 2018. Briefly, nuclei were extracted with a pestle in 50 µl ice-cold lysis buffer (10 mM Tris-HCl, pH 8, 10 mM NaCl, 0.2 % IGEPAL CA-630) and additional 150 µl ice-cold lysis buffer was added. From here, we followed the protocol as described in Ramírez et al. 2018 except for the following modifications: nuclei were digested for 3 hr in total using 2 µl DpnII (NEB, R0543M, 150 U/ sample), half of enzyme amount added after 1.5 hr. During ligation, the ligase (T4 DNA ligase, NEB 0202S) was also added in two steps. DNA purification was performed using the Zymo ChIP DNA Clean & Concentrator kit (D5205). To increase the fraction of valid Hi-C reads, dangling ends were removed using 3 U of T4 DNA polymerase for 30 min at 20 °C with addition of 100 µM dGTP and dATP. After biotin pull-down, all DNA bound to beads was used for library preparation and libraries were sequenced paired-end, with a read length of 75 bp. NEB Next Ultra II