GSM8946452: HCT116, untreated, biol replicate 3; Homo sapiens; OTHER
For each replicate, approximately 5 million cells were crosslinked in 2% formaldehyde and 10% FBS in PBS for 10 min while rotating at room temperature. Glycine was added to a final concentration of 0.125 M to quench crosslinking reaction and cells were centrifuged at 500 x g for 5 min. Cells were washed twice with PBS, transferred to an Eppendorf tube, and lysed on ice for 20 min with 1 mL lysis buffer (50 mM Tris-HCl (pH 7.5), 10 mM NaCl, 5 mM EDTA, 0.5% NP-40 and 1% Triton X-100) supplemented with 1X protease inhibitors (Roche). Cells were then washed once with 450 µL of 1.2X DpnII buffer and resuspended in 500 µL 1.2X DpnII buffer and 15 µL of 10% SDS (final: 0.3% SDS) and incubated in a thermoshaker at 37 °C for 1 h at 750 RPM. Then, 75 µL of 20% Triton X-100 (final: 2.5% Triton-X-100) were added, nuclear aggregates were resuspended with a pipet, and tubes were incubated at 37°C for 1 h while shaking at 750 RPM. A 5 µL aliquot of undigested control was stored, and nuclei were incubated overnight with 250 U of DpnII restriction enzyme (New England Biolabs). A fresh 250 U of DpnII was added the following morning, and samples were digested for an additional 3 h. After this, digestion was checked for completion by running 5 μl of sample on a 0.6% agarose gel next to undigested lysates. DpnII was inactivated by incubating the reaction tubes for 20 min at 65 °C, and a proximity ligation reaction was performed in a 7 mL volume with 3200 U of T4 DNA ligase (New England Biolabs) for 16 h at room temperature followed by an overnight period at 16 °C and finally a 500 U T4 DNA ligase spike-in and another 36 h incubation at room temperature. After this, ligation was checked for completion by running 5 μl of sample on a 0.6% agarose gel next to the digested control. Cross-links were reversed at 65 °C overnight after adding 300 μg proteinase K. Samples were then treated with 300 μg RNase A for 30 min at 37 °C, and DNA was purified using MagMax DNA binding beads (Thermo Fisher Scientific) and a magnetic bead-based protocol described in a previous reference 50. A second restriction digest was performed overnight at 37 °C while shaking at 500 RPM in a 500 μL reaction with 200 U of NlaIII (New England Biolabs). After this, digestion was checked for completion by running 5 μl of sample on a 0.6% agarose gel next to the samples from the first ligation. NlaIII was inactivated at 65 °C for 25 min, and a proximity ligation reaction (5 ng/μL DNA concentration) was performed in a 5 mL volume with 2000 U of T4 DNA ligase for 16 h at room temperature followed by an overnight period at 16 °C and finally a 400 U T4 DNA ligase spike-in and another 36 h incubation at room temperature. After ligation, DNA was again purified using MagMax DNA binding beads (Thermo Fisher Scientific) To generate the 4C–seq library, several viewpoint-specific inverse PCR primer sets targeting the MYC promoter conserved CTCF binding site were compared for efficiency and the primer pair that produced the strongest product was selected for the 1st round PCR in library preparation. 200 ng of prepared 4C template was amplified with 16 PCR cycles in 4 separate 50 μL reactions using Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs). Next, these 4 reactions were mixed and 50 μL of 1st round PCR product was purified using ChargeSwitch PCR Clean- Up Kit (Thermo Fisher Scientific) to remove primer dimers. 5 μL purified 1st round PCR product was used as a template in a 2nd round of PCR for 20 cycles in a 50 μL reaction using Q5 Hot Start High-Fidelity DNA Polymerase (New England Biolabs) to add Illumina P5/P7 flow cell adapters, Illumina sequencing primer sites, and unique 6 bp indexes to all replicates. These 2nd round reactions were then purified with the QIAquick PCR purification kit (QIAGEN), the purity was measured with a Nanodrop 2000 Spectrophotometer (Thermo Fisher Scientific), the concentration was more accurately determined using the Qubit dsDNA HS Assay (Thermo Fisher Scientific), and all replicates were run on the Bioanalyzer 2100 (Agilent) to check for any remaining primer dimers and determine average library size. After final quality control and Element library circularization, the 4C-seq libraries were sequenced on the Element Biosciences AVITI for 150 bases in paired-end read mode at 300 cycles.