title: GSM3097127: HiC_siTIP5_1; Mus musculus; OTHER
library_construction_protocol: HiC experiments were performed in triplicates of ESC+2i treated with siRNA-control or siRNA-TIP5. Five million cells were pelleted and resuspended in PBS-10%FCS. PBS-10%FCS-4% formaldehyde was added to a final concentration of 2% formaldehyde (v/v). Samples were incubated at room temperature for 10 minutes with mixing. Ice-cold glycine solution was added to a final concentration of 0.2M and immediately centrifuged for 5 minutes at 300xg at 4°C. Cells were washed in 1 ml of ice-cold PBS. Pellet was flash-frozen in liquid nitrogen. Pellet was taken up and washed in 1 ml ice cold lysis buffer1, resupended again in 1 ml of ice cold lysis buffer and incubated for 30 minutes at 4°C.  Sample was pelleted and washed in 0.5 ml 1.2x DpnII buffer, resuspended in 0.5 ml of DpnII buffer again and moved to a thermomixer at 37°C and 300 rpm. SDS was then carefully added to a concentration of 0.3%, slowly suspended with a pipet and incubated for an hour. Triton X-100 was added to a concentration of 2.6% and sample was incubated for another hour. To digest the sample, 200 units of DpnII enzyme were added for a 4 hour incubation in a thermomixer at 37°C and 900 rpm; another 200 units of DpnII were added for overnight incubation. From here on, we adopted the protocol described in (Rao et al., 2014) with some adjustments Cells were incubated for 20 minutes at 65°C to heat inactivate DpnII, pelleted and 300 ul of fill-in mastermix was added (218 μl of MilliQ, 30 μl of 10x NEB buffer 2, 15 μl of 10mM dCTP, 15 μl of 10mM dGTP, 15 μl of 10mM dTTP, 37.5 μl of 0.4mM biotin-14-dATP (Life Technologies, 19524-016), 10 μl of 5U/μl DNA Polymerase I, Large (Klenow) Fragment (NEB, M0210)). Sample was mixed by pipetting and incubated for 60 minutes @ 37°C shaking at 300 rpm and placed at 4°C afterwards. 900 ul of ligation mix was added (120 ul 10x ligase buffer, 50 units of T4 ligase (Roche) and 770 ul MilliQ) and mixed by inverting and incubated overnight at 16°C. Sample was pelleted for 5 minutes at 1000 x g and taken up in 500 µl 10 mM Tris. Protein was degraded by adding 50 μl of 20mg/ml proteinase K (NEB, P8102), 50 μl of 10% SDS and incubated at 55°C for 30 minutes. 57 μl of 5M of sodium chloride was then added and the sample was incubated at 68°C overnight or for at least 1.5 hours. Samples were cooled to room temperature and DNA was purified using NucleoMag P-Beads and taken up in 5mM Tris pH7.5. Samples were sheared to a size of 300-500 bp using a Covaris S2 focused-ultrasonicator. From here on, the protocol described in (Rao et al., 2014) was adopted.
attributes.tag: GEO Accession
attributes.value: GSM3097127