GSM4879712: WT mESCs - TINC-seq Nanog locus TALE 1 rep2; Mus musculus; OTHER
Approximately 1 x 10^9 cells were fixed with formaldehyde and nuclei and chromatin were isolated to remove non-chromatin-associated proteins. The chromatin was then resuspended in ChIP lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH 8) containing protease inhibitor cocktail (Sigma Cat#P3840) at 1g of chromatin per 3.2mL of buffer and sonicated on a Bioruptor NextGen device (Diagenode) to shear the chromatin to an average fragment size of 200 - 500 bp. The samples were then diluted 1:5 with ChIP dilution buffer (165 mM NaCl, 0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8.0) containing protease inhibitor cocktail and 0.05% input was put aside on ice. Additionally, 0.05% of the sample was used to determine the DNA concentration to ensure the same amount of chromatin was used for TALE and control samples in the TINC assay. To do so, cross-linking was reversed for 15 min at 100°C, then the sample was purified using a QIAquick PCR Purification Kit (Qiagen) and DNA concentration determined using the Qubit dsDNA HS Assay Kit and Qubit 2.0 Fluorometer (Thermo Fisher Scientific). The samples were then precleared with 1 mL of settled Control Agarose Resin (Pierce Cat#26150) for 1h at 4°C.  TALE-immunoprecipitations were performed with 1 mL of settled Anti-HA Agarose Resin (Pierce 26182) overnight at 4°C. Samples were then washed twice for 10 min at 4°C with dilution buffer containing protease inhibitor cocktail, followed by two washes for 10 min at 4 °C with low salt buffer (150 mM NaCl, 0.5% Na deoxycholate, 0.1% SDS, 1% NP-40, 1 mM EDTA, 50 mM Tris pH 8) containing protease inhibitor cocktail, two washes for 10 min at 4°C with high salt buffer (500 mM NaCl, 0.5% Na deoxycholate, 0.1% SDS, 1% Nonidet P-40, 1 mM EDTA, 50mM Tris pH 8) and a final 5 min wash at 4 °C with TE buffer (0.25 mM EDTA, 10mM Tris pH 8). The volume was 100 mL for each wash step. For elution, the samples were resuspended in 2 mL of 3M NaSCN and incubated rotating for 5 min at room temperature. The eluted samples were removed and the elution step was repeated three times. All four elutions were pooled and concentrated using 3 KDa cut-off concentrators (Amicon Ultra, Cat#UFC800308 and Cat#UFC500308) to 50 μL. During concentration, the samples were diluted 1:20 with PBS to dilute the NaSCN. 1 μL per sample was then removed and processed along with the input sample for DNA purification. Libraries were prepared using the ThruPLEX DNA-seq 12S kit (Rubicon Genomics, Cat#R400429). After limited PCR amplification, libraries were purified using Agencourt AMPure XP beads (Beckman Coulter, Cat#A63880), according to the manufacturer's recommendations. TINC-seq samples 75 bp single reads were sequenced on a NextSeq550 High output mode and v2 chemistry.