title: GSM5915088: AUX_TRP_3; Homo sapiens; OTHER
library_construction_protocol: Hi-C was performed largely as described in Rao et al, 2014 with minor modifications. Briefly, ~5x10 6 HCT116 cells were crosslinked in 1% formaldehyde for 10 min at RT, snap-frozen and stored at -80°C. After permeabilization in lysis buffer (0.2% Igepal, 10 mM Tris-HCl pH 8.0, 10 mM NaCl, 1x Halt Protease inhibitor cocktail) nuclei were isolated in 0.3% SDS in NEBuffer 3 at 62°C for 10 min. SDS was quenched with 1% Triton X-100 at 37°C for 1 h, then the nuclei were pelleted and resuspended in 250 μl DpnII buffer with 600 U DpnII (New England Biolabs) at 37˚C. After overnight digestion, 200 U DpnII were added for 2 h more incubation. Then, the ends were filled-in with d(G/C/T)TPs and biotin-14-dATP (Life Technologies) using Klenow (New England Biolabs) for 1.5 h at 37°C. After ligation at room temperature for 4 h with T4 ligase (New England Biolabs), the nuclei were spun down, resuspended in 200 μl mQ and digested with proteinase K for 30 min at 55°C in presence of 1% SDS. Cross-links were reversed at 65°C overnight after addition of NaCl to a final concentration of 1.85 M. After ethanol precipitation and a 70%–80% ethanol wash, DNA was resuspended in 50 µl of 10 mM Tris EDTA, incubated on ice for 15 min and then sheared to ~300-1300 bp fragments using the E220 Focused-ultrasonicator (Covaris). DNA was bound to MyOne C1 Streptavidin beads (ThermoFischer) and used for library preparation with the NEB Next Ultra Library Prep kit (Illumina). Micro-C was performed using the Dovetail micro-C kit. Libraries were sequenced on the NovaSeq 6000 system with 100 bp paired-end reads (at VBCF NGS Unit) The library preparation was done by the VBCF NGS unit (vbcf.ac.at) or MPIB NGS Core facility (https://www.biochem.mpg.de/7076201/NGS) following the standard DNA sample preparation protocol.