GSM4790415: OSC - Piwi-KD - HiC_rep1; Drosophila melanogaster; Hi-C
Hi-C: Hi-C using OSCs were performed as described previously (Ulianov et al., 2016) with some minor modifications. OSCs were fixed with 1% formaldehyde for 10min, and stored at -80 degrees before usage. 5 x 10^5 cells were lysed and chromatin digested using 30G needle. Chromatin digestion was performed using HindIII restriction enzyme. Ends were filled with biotin-14-dCTP followed by blunt-end ligation. Proteins were reverse crosslinked form the cells, and DNA purification was performed. Biotin was removed from un-ligated ends and DNA was sonicated using covaris S220 (covaris). Sonicated DNA was used for biotin pull down by streptavidin conjugated to Dynabeads Streptavidin C1 beads (Invitrogen). Pulled-down DNA was used as the template to construct DNA library. DamID-seq: Dam-ID-seq was performed as described previously (Pickersgill et al., 2006) with some modifications to analyze methylated sequences using deep-sequencing rather than microarray. DamID vectors were transfected into OSCs using Nucleofector, one day before cell harvest. pNDamMyc plasmid (van Steensel and Henikoff, 2000) was a kind gift from Dr. Bas van Steensel. Lamin was cloned into pNDamMyc vector as described previously (Pickersgill et al., 2006). Genome DNA precipitation, DpnI digestion, adapter ligation, DpnII digestion, PCR, and PCR cleanup was performed as described within “Van Steensel Lab version 28/08/2006” protocol. Obtained DNA was used as the template to prepare deep sequencing library. ChIP-seq: ChIP was performed as described previously (Iwasaki et al., 2016). Briefly, OSCs (3×107) were fixed, lysed, and their nuclei were isolated using truChIP Chromatin Shearing Kits (Covaris), in accordance with the manufacturer's instructions. DNA was sonicated to ~300 bases using Bioruptor (Cosmobio), and then diluted with ChIP buffer. DNA–protein complexes were incubated with antibody on Dynabeads-Protein G for 4 h, at 4 degrees. Beads were washed with ChIP buffer, high-salt lysis buffer, and wash buffer followed by TE. After reversing cross-linking for 12–16 h at 65 degrees, samples were treated with RNase for 30 min at 37 degrees and Proteinase K for 60 min at 55 degrees. DNA was then recovered. DNA library was constructed using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB), in accordance with the manufacturer's instructions.