title: GSM9328759: HCT116-GFP, NoTriptolide, Rep2 (EnChAMP-seq); Homo sapiens; OTHER
library_construction_protocol: Cells were harvested at ~90% confluency (~20 million cells/15cm plate). Cells were washed with DPBS, scraped, and collected by centrifugation at 800g for 5 minutes at 4°C. Supernatants were discarded. Nuclei Isolation: Cell pellets were resuspended in 1 mL of ice-cold Hi-C Buffer A (10 mM HEPES [pH 7.5], 10 mM KCl, 1.5 mM MgCl2, 1 mM CaCl2, 0.1% Digitonin (Millipore Sigma, Cat# 300410), with 1X Phosphatase (Sigma Aldrich, Cat# P2850) and 1X Protease inhibitor cocktails (Thermo Fisher Scientific, Cat# A32965), and 1 mM PMSF (Cell Signalling, Cat# 8553S)) and incubated on ice for 30 minutes to allow cell swelling. After incubation, aliquots were taken for western blot analysis. The remaining lysates were centrifuged at 1000g for 15 minutes at 4°C. Nuclei pellets were washed with 1 mL of cold Hi-C Wash Buffer B (Hi-C Buffer A supplemented with 250 mM sucrose), centrifuged as above, and supernatants containing cytoplasmic fraction was discarded. Chromatin Isolation: Nuclei pellets were resuspended in 500 μL of 1.5% Digitonin Chromatin Isolation Buffer A (10 mM HEPES [pH 7.5], 20 mM KCl, 150 mM NaCl, 1.5 mM MgCl2, 1 mM CaCl2, 15% Glycerol, 1.5% Digitonin, with phosphatase and protease inhibitors, and PMSF) and incubated on ice for 30 minutes. Samples were centrifuged at 1000g for 30 minutes at 4°C, and supernatants containing nucleoplasmic fraction were discarded. The pellets were resuspended in 500 μL of fresh Buffer A, and 3 μL of 250 U/μL Benzonase® Nuclease (Millipore Sigma, Cat# E1014) was added for chromatin shearing. The samples were rotated at 4°C for 1 hour, followed by centrifugation at 1000g for 30 minutes at 4°C. Soluble chromatin proteins were collected as supernatants, while the insoluble chromatin fractions were discarded. Affinity purification with anti-GFP nanobody: During Benzonase digestion of chromatin, per sample 10 μL of GFP-Trap® M-270 magnetic particles (ChromoTek, Cat#gtd) were washed three times with Chromatin Isolation Buffer B (10 mM HEPES [pH 7.5], 20 mM KCl, 150 mM NaCl, 1.5 mM MgCl2, 1 mM CaCl2, 15% Glycerol) and resuspend in 50 μL of Buffer B. Soluble chromatin proteins (~500 μL) were incubated with 50 μL of pre-washed magnetic particles per sample overnight at 4°C. Beads were washed sequentially with Chromatin Isolation Buffer B: a 5-minute rotation wash, followed by two stationary washes, and a final transfer to a clean tube. Beads were eluted with 200 μL of 100 mM HEPES (pH 7.5) containing 1% SDS at 65°C for 15 minutes. The supernatants (eluted chromatin) were collected for downstream analysis. Eluates  of ~5M cell equivalent from EnChAMP experiments were brought to 300ul with 1X TE buffer (10 mM Tris-Cl pH 8.0, 0.1 mM EDTA), extracted once with equal volume Phenol:Chloroform mix pH 8.0 (Thermo Fisher Scientific, Cat# 17909), extracted with equal volume Chloroform, EtOH precipitated (1/10th volume 3M NaAcetate pH 5.2, 2 μl GlycoBlue  co-precipitant (Thermo Fisher Scientific, Cat# AM9516), and 3 volumes 100% EtOH, washed with 70% EtOH, and air dried pellet was resuspended in MilliQ-H2O. DNA amount was quantitated by Qubit dsDNA HS Assay (Thermo Fisher Scientific, Cat# Q32851). 2-40 ng DNA was end-repaired in the presence of a 250 μM dNTP mix (each), 1x T4 DNA Ligase Buffer (NEB), 6 units T4 DNA Polymerase (NEB, Cat# M0203L), 20 units T4 PNK (NEB, Cat# M0201L), and 2.5 units Klenow Polymerase (NEB, Cat# M0210L) at RT for 30 min on a Thermomixer at 600 rpm. End-repaired DNA was purified using MinElute Reaction Clean-up Kit (Qiagen, Cat# 28204) and A-tailed in the presence of 1x Buffer #2 (NEB), 200 uM dATP, and 10 units Klenow exo- polymerase (NEB, Cat# M0212L) at 37C for 30 min on a Thermomixer at 600 rpm. A-tailed DNA was purified using MinElute Reaction Clean-up Kit (Qiagen, Cat# 28204) and ligated to Illumina TRUseq adapters in the presence of 12-120 nM TRUseq Index adapter, 1x T4 DNA Ligase buffer (NEB), 1,200 units T4 DNA Ligase (NEB, Cat# M0202L) at 18 ºC O/N. TRUseq adapter ligated DNA was purified using MinElute Reaction Clean-up Kit (Qiagen) and subjected to a pilot PCR using P5 and P7 oligos to determine appropriate number of PCR cycles to generate a library. Final PCR reactions were carried 11-15 cycles and purified using MinElute PCR Clean-up Kit (Qiagen, Cat# 28004). After quality check with BioAnalyzer analysis, if deemed necessary the libraries were gel purified (150-350 bp region) using 8% native PAGE to get rid of the adapter dimer (~130 bp). Equal amounts of 8-12 samples were pooled and sequenced either by 2x150nt on an Illumina NovaSeq 6000 instrument (NovaGene) or by 2x80nt on an Element Biosciences AVITI instrument (EGC Core Facility).