GSM9598952: Kc167, mock cell, bio rep1; Drosophila melanogaster; Hi-C
Hi-C libraries were preared following the optimized Hi-C 2.0 protocol (Belaghzal et al., 2017) anad adapted for Drosophila cells. 4-5x10^7 cells per condition were crosslinked with 1% fromaldehyde and quenched with 0.125M glycine. Cells were washed with cold PBS, snap-frozen, and stored at -80C until further processing. Cells were lysed in cold lysis buffer and chromatin from isolated nuclei were permeabilized with 1x NEBuffer 3.1 containing 0.1% SDS and quenched with 1% Triton X-100. DNA was digested with DpnII (NEB) and 5' overhangs were were filled using Klenow DNA polymerase (NEB) and labeled with biotin-14-dATP. Proximity blunt-end ligation was performed in situ using T4 DNA ligase (Invitrogen) followed by Proteinase K treatment to reverse crosslinks. DNA was purified by repeated phenol:chloroform extraction followed by ethanol precipitation and concentration using 30 kDa Amicon columns. Biotin was removed from unligated ends using T4 DNA polymerase (NEB) in the presence of dATP and dGTP (Invitrogen). Free nucleotides including biotin-14-dATP were removed by repetitive concentration with Amicon columns. DNA was then sheared to ~200–300 bp fragments using the Bioruptor (Diagenode). Biotinylated ligation junctions were captured using Dynabeads MyOne Streptavidin C1 (Invitrogen). Sequencing libraries were prepared using the TruSeq ChIP Library Preparation Kit (Illumina), and AMPure XP beads (Beckman Coulter) selection was used to remove adapter and primer dimers.