Instrument: Illumina NovaSeq 6000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: ZIP13K2-derived EN cells (5 x 106 /IP) were harvested and cross-linked in 1% formaldehyde (Thermo Fisher Scientific, 28908) in DPBS for 10 min at RT, followed by quenching with final 125 mM Glycine (Sigma-Aldrich, 50046) for 5 min at RT. Cross-linked cells were then centrifuged at 500 x g at 4°C and washed twice with ice cold DPBS. Cell lysis was performed by resuspending the pellet in 500 μl Cell Lysis Buffer (Final 10mM Tris-HCl, pH 8,0 (Sigma Aldrich, T2694); 85mM KCl (Sigma Aldrich, P9541); 0,5% NP40 (Sigma Aldrich, 56741); 1 x cOmplete, EDTA-free Protease Inhibitor Cocktail (Sigma Aldrich, 11873580001)) followed by 10 min incubation on ice. After the incubation, lysed cells were centrifuged at 2500 x g for 5 min at 4°C. Supernatant was carefully removed and the extracted nuclei were then resuspended in 230 μl Nuclei Lysis Buffer (Final 10mM Tris-HCl, pH 7,5 (Sigma Aldrich, T2319)); 1% NP40; 0,5% sodium deoxycholate (Sigma Aldrich, D6750); 0,1% SDS (Thermo Fisher Scientific, AM9820); 1 x cOmplete, EDTA-free Protease Inhibitor Cocktail). Following 10 min incubation on ice, each 260 μl sample was split into two microTUBEs (Covaris, 520045) and chromatin was sonicated using a Covaris E220 Evolution with the following settings: Temperature à 4°C; Peak power à 140; Duty factor à 5,0; Cycles/Burst à 200; Duration à 750 sec. After sonication, sheared chromatin (ranging from 200-600bp) was transferred in a new 1,5 ml tube and centrifuged at max speed for 10 min at 4°C. Supernatant was then transferred into a new tube and volume was increased to 1 ml /sample with ChIP Dilution Buffer (Final 16,7mM Tris-HCl, pH 8,0; 1,2mM EDTA (Sigma Aldrich, 03690)); 167mM NaCl (Sigma Aldrich); 1,1% Triton-X (Sigma Aldrich); 0,01% SDS; 1 x Protease Inhibitor). 50μl (5%) was then transferred into a new tube and frozen at -20°C as INPUT. 1μg of SOX17 antibody /106 initial cells was added to the 950 μl left, and immunoprecipitation was carried out at 4°C o/n on a rotator (s. Suppl. Table 3). The next day, 50μl of Dynabeads Protein G (Thermo Fisher Scientific, 10004D) /IP were washed twice with ice cold ChIP Dilution Buffer and then added to each IPs. IP/bead mixes were incubated for 4 hours at 4°C on a rotor. Next, bead/chromatin complexes were washed twice with Low Salt Wash Buffer at 4°C (Final 20 mM Tris-HCl, pH 8,0; 2 mM EDTA; 150 mM NaCl (Sigma-Aldrich, S6546); 1% Triton-X; 0,1% SDS), twice with High Salt Wash Buffer at 4°C (Final 20 mM Tris-HCl, pH 8,0; 2 mM EDTA; 500 mM NaCl; 1% Triton-X; 0,1% SDS), twice with LiCl Wash Buffer at 4°C (Final 10 mM Tris-HCl, pH 8,0; 1mM EDTA; 250mM LiCl (Sigma Aldrich, L9650); 1% sodium deoxycholate (Sigma Aldrich); 1% NP40), twice with TE pH 8,0 (Sigma Aldrich, 8890) at room temperature and finally eluted twice in 50 μl freshly prepared ChIP Elution Buffer (Final 0,5% SDS; 100 mM NaHCO3 (Sigma Aldrich, S5761)) at 65°C for 15 min (total 100 μl final eluent). Thawed INPUTS and eluted IPs were next reverse cross-linked at 65°C o/n after the addition of 16 ul freshly prepared Reverse Crosslinking Salt Mixture (Final 250 mM Tris-HCl, pH 6,5 (Sigma Aldrich, 20-160); 62,5 mM EDTA; 1,25M NaCl; 5 mg/ml Proteinase K (Thermo Fisher Scientific, AM2548)). The following day phenol:chloroform (Thermo Fisher Scientific, 15593031) extraction followed by precipitation was performed to isolate DNA. IPs and INPUTS were then quantified and NGS libraries were prepared using NEBNext Ultra II DNA Library Prep Kit for Illumina (New England Biolabs, #E7645) following the manufacturer's instructions. Library quality and size distribution was verified using a TapeStation D5000 HS kit (Agilent Technologies, 5067-5592). Samples were sequenced with a coverage of 50 M paired end reads (2 x 100 bp) /sample on a NovaSeq (Illumina).