title: GSM2256195: 4C_hela_myc_r1; Homo sapiens; OTHER
library_construction_protocol: Single cell suspensions consisting of 5M cells per sample were prepared by dissociating cells with Trypsin/EDTA, centrifuging at 300g for 5 min, and resuspending in 10% (vol/vol) FBS/PBS. Cells were fixed by adding 37% formaldehyde to a final concentration of 1% and incubating for 10 min at room temperature while tumbling, followed by addition of 1M glycine to a final concentration of 0.125M to quench. Samples were centrifuged at 300g for 8 min at 4°C, and supernatant was removed. Samples were washed with cold PBS and resuspended in 5 mL ice-cold lysis buffer (10 mM Tris-HCl (pH 8.0), 10 mM NaCl, 0.2% NP-40, 1× complete protease inhibitor (Roche)), followed by 10 min incubation on ice. Cytoplasmic lysis was then confirmed by light microscopy. Samples were centrifuged at 300g for 5 min at 4°C and resuspended in 500 uL 1.2x CutSmart restriction digest buffer (NEB). 20% SDS was added to a final concentration of 0.3%, and samples were incubated for 1 hr at 37°C while shaking at 900 rpm on a thermomixer. 20% Triton X-100 was added to a final concentration of 2%, followed by incubation for 1 hr at 37°C while shaking at 900 rpm on a thermomixer. 600U of NlaIII (NEB) was added to each sample and incubated 20 hr at 37°C while shaking at 900 rpm on a thermomixer. To inactivate enzyme, 20% SDS was added to a final concentration of 1.6% SDS, followed by incubation for 25 min at 65°C while shaking at 900 rpm on a thermomixer. Samples were added to 6.125 mL of 1.15x T4 DNA Ligase buffer (NEB) and 10,000 U of T4 DNA Ligase (NEB), followed by incubation for 18 hr at 16°C. De-crosslinking was performed by adding 300 ug proteinase K (Machery Nagel) and incubating for 16 hr at 65°C. 300 ug RNase (Sigma) was added and incubated for 30 min at 37°C. 7 mL phenol:chloroform was added to each sample and centrifuged at 3,200g for 15 min. Aqueous phase was transferred to 7mL H2O, 1.5 mL 2M NaOAc (pH 5.6), 7 uL glycogen, and 35 mL 100% ethanol. Mixtures were frozen at -80°C for 2 hrs, until solid, and immediately centrifuged at 3,200g for 45 min at 4°C. Pellets were washed with 70% ethanol and centrifuged at 3,200g for 15 min at 4°C. Pellets were air dried and resuspended in 150 uL 10 mM Tris-HCl (pH 7.5). The second 4C-seq digest was then performed by adding 50 U DpnII (NEB), 50 uL 10x NEBuffer DpnII, and H2O to 500 uL, followed by incubation for 16 hr at 37°C. Digests were then purified using phenol:chloroform and resuspended in 100 uL H2O. Second ligation was performed by transferring samples to 12.5 mL H2O, 1.4 mL 10x T4 DNA Ligase buffer (NEB), and 20,000 U of T4 DNA Ligase (NEB), followed by incubation for 18 hr at 16°C. Circularized fragments were purified by phenol:chloroform and resuspended in 75 uL 10 mM Tris-HCl (pH 7.5), and then purified again using NucleoSpin PCR Clean-up (Machery Nagel), distributing each sample across 3 columns. 1.2 ug of each template was amplified using the Expand Long Template PCR kit (Roche) for 30 cycles. PCR primers used were selected from a genome-wide database generated previously: MYC viewpoint Forward – TTGCACTTTTCTTGTCCATG; MYC viewpoint Reverse – GGGGAAGGAATAAAAGAAAA . PCR amplicons were purified using Agencourt Ampure XP beads. Sequencing libraries were generated using the KAPA HyperPlus kit according to manufacturer’s recommendations, with the following modifications: fragmentation was carried out for 20 min, and adapter ligation was performed using 10 uL of 15 uM illumina adapters, incubated for 75 min at 20°C. No further amplification was performed following ligation. Kapa HyperPlus