title: GSM6568205: MYCN TSC replicate3; Mus musculus; OTHER
library_name: GSM6568205
library_construction_protocol: Briefly, 10 million cultured ESC, TSC and XEN cells were fixed with 12 ml of 1% formaldehyde (Thermo Scientific, 28908) in 10% FBS for 10 min at room temperature (RT) (tumbling). Quenching of the cross-linking was performed with the addition of 1.8 ml of freshly prepared ice-cold 1 M glycine (Sigma-Aldrich #500046). Tubes were transferred directly on ice and centrifuged for 5 min 500g at 4°C. Cells were washed with 1× PBS and centrifuged for 5 min 500g at 4°C, and pellets were frozen in liquid nitrogen and stored at −80°C. Next, cells were then vigorously resuspended in 1 ml of fresh ice-cold lysis buffer (10 mM tris (pH 8), 10 mM NaCl, 0.2% NP-40, and 1 tablet of complete protease inhibitor (Roche, 04693159001)], transferred to 9 ml of prechilled lysis buffer, and incubated for 20 min on ice. Following centrifugation at 500g for 5 min at 4°C, the pellet was resuspended in 50uL of 0.5% SDS and incubated for 10 min at 65°C. SDS was quenched with 145uL ddH2O and 25uL of 10% Triton X-100 for 15 mins at 37°C. At this point, 5 l of the sample was taken as the “undigested control”. Next, 25ul of CutSmart buffer (NEB, B7204S) was added with 10μl DpnII enzyme (NEB, R0543M) and the samples were incubated overnight at 37°C under agitation (750rpm). Upon first digestion, 5μl of the sample was taken as the “digested control” while the efficiency of chromatin digestion was verified after DNA extraction from 5l of undigested and digested controls and loading in a 1.5% agarose gel. After verification of chromatin digestion (smear between 0.2 and 2 kb), DpnII was deactivated by 20 min incubation at 62°C (under agitation 750 rpm). Ligation of DNA ends between the cross-linked DNA fragments was performed  by diluting the samples in 669 μL ddH2O and adding 120 μL T4 ligation buffer (NEB, B0202), 60 μL 10mM ATP (NEB, P0756S), 120 μL 10% Triton X-100, 6 μL 20mg/ml BSA and 5 μL 400U/μl T4 DNA Ligase (NEB, M0202) overnight at 16oC (tumbling)  followed by 30min at RT. 10μl  of the ligated sample was tested as “ligated control,” on a 1.5% agarose gel. The samples were then treated with proteinase K and reverse crosslinked overnight at 65oC. Following RNase treatment, phenol/chloroform extraction and DNA precipitation, the pellets were dissolved in 100 μL of 10mM Tris pH 8 and incubated for 1 hour at 37°C. Efficiency of extraction and purification were verified on a 1.5% agarose gel. For the second digestion 20 μL of 10x buffer B (Fermentas), 10 μL Csp6I (Fermentas, ER0211), 80 μL ddH2O were added to the DpnII-ligated 3C template and samples were incubated overnight at 37°C under agitation (750rpm). Csp6I was inactivated at 65°C for 20 min, and DNA fragmentation was tested on 1.5% agarose gel. A second ligation was performed by adding 300 μL T4 ligation buffer, 150 μL 10mM ATP, 5μL T4 DNA Ligase, and ddH2O to 3mL and incubating overnight at 16°C. After 30 min of incubation at RT, samples were PCI-extracted, ethanol-precipitated, resuspended in 200 l of sterile water, and purified using the Qiaquick PCR Purification Kit (Qiagen). DNA concentration of each digested sample was calculated using the Qubit brDNA HS assay kit (Invitrogen). For library preparation, primers were designed either around the enhancer or the promoter of lineage specific genes. Library preparation was then performed using the inverse PCR strategy. Briefly, 4x200 ng of 4C-template DNA was used to PCR amplify the libraries using the Roche Expand long template PCR system (Roche, 11681842001) with the following PCR conditions: 94 °C for 2 min, 16 cycles: 94 °C for 10 s; [primer specific]°C for 1 min; 68 °C for 3 min, followed by a final step of 68 °C for 5 min. Amplified material was pooled, and primers were removed using SPRIselect beads (Beckman Coulter, B23317). A second round of PCR with the following conditions: (94 °C for 2 min, 20 cycles: 94 °C for 10 s; 60 °C for 1 min; 68 °C for 3 min and 68 °C for 5 min) was performed using the initial PCR library as a template, with overlapping primers to add the P5/P7 sequencing primers and indexes.