title: GSM8745079: NPC, CTCF-ΔZF1 rescue, rep1; Mus musculus; OTHER
library_construction_protocol: Cells in a confluent 15 cm tissue culture plate (~30 million (M) cells) were dissociated with TrypLE Express and crosslinked with freshly made 3 mM ethylene glycol bis (EGS) in PBS (5 mL per 5M cells), incubated at room temperature for 45 minutes. Crosslinking reaction was quenched by adding Tris pH 7.5 to final 0.75 M concentration and incubated at room temperature for 5 minutes. EGS-crosslinked cells were washed twice with 0.5% BSA in PBS and subjected to the second crosslinking with 1% formaldehyde in PBS (5 mL per 5M cells), incubated at room temperature for 10 minutes. Crosslinking reaction was quenched again by adding Tris pH 7.5 to final 0.75 M concentration, incubated at room temperature for 5 minutes. Cells were washed twice with 0.5% BSA in PBS and aliquoted into 5M cells per 1.5 mL tube. Cell pellets were spun down, snap-frozen, and stored in −80°C. Crosslinked cells (5M in 1.5 mL tubes) were thawed on ice and permeabilized in cold Micro-C Buffer (MB) #1 (50mM NaCl, 10mM Tris pH 7.5, 5mM MgCl2, 1mM CaCl2, 0.2% NP-40, 1x Protease Inhibitor Cocktail) for 20 minutes on ice. Chromatin from permeabilized cells was digested by adding micrococcal nuclease (MNase) (NEB M0247SVIAL) at 1:100 dilution and incubated at 37°C for 10 minutes, shaking at 850 rpm. Dilution of MNase was pre-titrated such that chromatin is digested efficiently into nucleosome-sized fragments. Enzyme activity was stopped by adding EGTA to a final concentration of 4 mM and incubated at 65°C for 10 minutes. MNase-digested chromatin was washed twice with ice-cold MB #2 (50mM NaCl, 10mM Tris pH 7.5, 10mM MgCl2). Digested chromatin fragments were then subjected to 3'-dephosphorylation and 5'-phosphorylation by 0.5 U/uL T4 Polynucleotide Kinase in 1x NEBuffer 2.1 (NEB B6002S) with 2 mM ATP and 5 mM DTT, incubated for 15 minutes at 37°C with interval mixing. 3'-end-chewing was done by adding 0.5 U/uL DNA Polymerase I Klenow Fragment and incubating another 15 minutes in 37°C. Blunt-end reaction was triggered by adding biotin-dATP, biotin-dCTP, dGTP, and dTTP to a final concentration of 66 uM and incubated at 25°C for 45 minutes. The reaction was stopped by adding 30 mM EDTA final concentration and incubating at 65°C for 20 minutes. Chromatin was washed once with cold MB #3 (50mM Tris pH 7.5, 10mM MgCl2). Chromatin fragments with biotin-dNTPs were then ligated with 20 U/uL T4 DNA ligase (NEB M0202M), incubated at room temperature for at least 2.5 hours with slow rotation. Unligated ends containing biotin-dNTPs were then removed by 5 U/uL exonuclease III in 1x NEBuffer 1 (NEB B7001S), incubated at 37°C for 15 minutes. Chromatin was subjected to reverse crosslinking with 1 mg/mL Proteinase K (Sigma 3115879001), 1% SDS, and 100 ug/mL RNase A (ThermoFisher Scientific EN0531), incubated at 65°C overnight. DNA was extracted by adding 1x volume Phenol:Chloroform:Isoamyl Alcohol (25:24:1) solution (Thermofisher Scientific 15593049) and isolating aqueous phase after centrifugation. DNA was purified from the aqueous phase using a PCR purification kit (Zymo Research DCC-5 D4013) following manufacturer's instructions. Separation of monomers and dimers was done by running purified DNA on 3% TBE agarose gel. 200-400 bp DNA was then extracted from the gel by Zymo Gel DNA recovery kit (Zymo Research D4008). DNA with biotin-dNTPs was captured by Dynabeads® MyOne Streptavidin C1 (ThermoFisher Scientific 65001). Standard library preparation protocol including end-repair, A-tailing, and adaptor ligation was performed on beads with enzymes from NEBNext Ultra II DNA Library prep kit for Illumina (E7645S). NEBNext Multiplex Oligos for Illumina® (Dual Index Primers Set 1, E7600S) were used for barcoding. Sequencing library was amplified by Kapa HiFi PCR enzyme (KAPA KK2601).