title: GSM5293830: K562_input; Homo sapiens; ChIP-Seq
library_construction_protocol: Approximately 1×107 GM12878 or K562 cells per sample were harvested, resuspended at a concentration of 1×106 per ml medium and crosslinked in 1% formaldehyde for 10 minutes at 37 ºC with mixing. The crosslinking reaction was quenched by the addition glycine at a final concentration of 125 mM and incubation for 5 minutes at room temperature. Cells were washed twice with PBS, centrifuged, and pellets were frozen at -80 ºC. Pellets were thawed on ice for at least 15 minutes, resuspended in ice-cold Hi-C Lysis Buffer (0.2% NP-40, 10 mM NaCl, 10 mM Tris-HCl, pH 8, complete protease inhibitor cocktail (Roche)), and incubated on ice for at least 15 minutes. Nuclei were centrifuged, the supernatant was discarded, and nuclei were washed with ice-cold Hi-C Lysis Buffer and centrifuged. Nuclei pellets were re-suspended in RIPA FA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, complete protease inhibitor cocktail (Roche)) and incubated for 10 minutes. Samples were sonicated with a Vibra-Cell VCX600 (Sonics & Materials) for 75 minutes total (2.2 seconds ON, 9.9 seconds OFF, 40% amplitude) to obtain DNA fragments with lengths averaging 150-350 nt (maximum 500 nt). After centrifugation at 20,000 g for 8 minutes, the supernatant was diluted 1:10 with Dilution Buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.1, 167 mM NaCl, complete protease inhibitor cocktail (Roche)) and aliquoted. Chromatin from an equivalent of 10×106 cells was used per immunoprecipitation reaction. For Lamin A/C immunoprecipitation, 104 µl of protein-A and 104 µl of protein-G Dynabeads (Invitrogen) were washed twice with RIPA buffer (0.1% deoxycholate, 0.1% SDS, 1% Triton X-100, 10 mM Tris-HCl, pH 8.1, 1 mM EDTA, 140 mM NaCl), resuspended in ChIP Blocking Buffer (PBS, 0.5% TWEEN, 0.5% BSA), and incubated with 50 µg of anti-Lamin A/C antibody (Santa-Cruz Biotechnology, sc-7292x) for at least 2 hours at 4 ºC with rotation. Sonicated chromatin was added to the conjugated beads, and samples were incubated for 16 hours at 4 ºC. The beads were washed six times with RIPA buffer, twice with RIPA-high salt buffer (0.1% deoxycholate, 0.1% SDS, 1% Triton X-100, 10 mM Tris-HCl, pH 8.1, 1 mM EDTA, 360 mM NaCl), twice with LiCl wash buffer (250 mM LiCl, 0.5% NP-40 (Sigma IGEPAL), 0.5% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1) and twice with TE buffer (10 mM Tris-HCl, pH 8.1, 1 mM EDTA). DNA was eluted from the beads with Elution buffer (0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris-HCl, pH 8.1) in a 30-minute incubation in a thermo-shaker at 65 ºC. From this stage on, input tubes were processed similarly to elution tubes. To the input and elution samples was added 1 µl of 10 mg/ml RNase A (Sigma), and samples were incubated for 30 minutes at 37 ºC. Next, 1.5 µl Proteinase K (NEB) was added, and samples were incubated for 16 h at 65 ºC. DNA was purified using phenol:chloroform:isoamyl extraction. DNA was eluted in 30 µl Elution buffer (10mM Tris-HCl pH 8.5). Concentrations were measured using a Qubit assay (ThermoFisher). ChIP-seq libraries were prepared for sequencing using standard Illumina protocols