title: GSM3660462: HiC_2.7kb_clone_replicate2; Mus musculus; Hi-C
library_construction_protocol: see library construction protocol 6x10^6 mESC were harvested and diluted in 1x PBS to final 1x106 cells/ml, then crosslinked with 1% formaldehyde and quenched with 0,125M glycine for 5 min at RT. After two 1x PBS washes, cells pellets were obtained by centrifugation, snap frozen and stored at -80°C. Pellets were thawed on ice and resuspended in 500 ul lysis buffer (10 nM Tris-HCl pH8.0, 10 nM NaCl, 0.2%NP40, 1x Roche protease inhibitors) and left 30 min on ice. Cells were then pelleted by centrifugation (954 x g, 5 min, 4°C ), washed once with 300 ul 1x NEB2 buffer and nuclei were extracted by 1 h incubation at 37°C in 190 ul 0.5%SDS 1xNEB2 buffer. SDS was neutralized by diluting the sample with 400 ul NEB2 buffer and adding 10% Triton X-100. After 15 min of incubation at 37°C, nuclei were pelleted, washed once in PBS and resuspended in 300 ul NEB2 buffer. 400U of MboI (NEB, 25 000 units/ml) were added and incubated at 37°C overnight. The next day, nuclei were pelleted again, resuspended in 200 ul fresh NEB2 buffer and additional 200U of MboI were added for two more hours before heat inactivation at 65°C for 15 min. 43 ul of end-repair mix (1.5 μL of 10 mM dCTP; 1.5 μL of 10mM dGTP; 1.5 μL of 10 mM dTTP; 37.5 μL of 0.4 mM Biotin-11-dATP (Invitrogen) and 1 μL of 50U/μL DNA Polymerase I Large Klenow fragment (NEB)) were added to the nuclear suspension, incubated at 37°C for 45 min and heat inactivated at 65°C for 15 min. The end repair mix was exchanged with 1.2 ml of ligation mix (120μL of 10X T4 DNA Ligase Buffer; 100 μL of 10% Triton X-100; 6 μL of 20 mg/mL BSA; 969μL of H2O) plus 5 ul of T4 ligase (NEB, 2000 units/ml) and ligation was performed at 16°C overnight. Nuclei were reconstituted in 200 ul fresh NEB2 buffer followed by RNA digestion in 0.5 mg/ml RNAse A for 10 min at 37°C. Samples were de-crosslinked with Proteinase K at 65°C overnight and DNA was purified using phenol/chloroform. 2 ug of DNA sample were sonicated using Diagenode Bioruptor Pico. MyOne Streptavidin T1 (Life Technologies # 65601) magnetic beads were used to capture biotinylated DNA followed by A-tailing. Adapter ligation was performed according to NEB Next Ultra DNA Library prep kit instructions. Two independent PCR reactions with multiplex oligos for Illumina sequencing were performed and pooled for the final PCR clean-up by magnetic AMPure bead (Beckman Coulter) purification. The final libraries were eluted in nuclease-free water, QCed by Bioanalyzer and Qubit
attributes.tag: GEO Accession
attributes.value: GSM3660462