title: SP112
organism: Homo sapiens
description: Protocols: ChIP-seq was performed using FUCCI-Human Embryonic Stem Cells (FUCCI-hESCs, H9 from WiCell) in a modified endoderm differentiation protocol (see details below). Cells were grown in defined culture conditions as described previously (Brons et al 2017). Pluripotent cells were maintained in Chemically Defined Media with BSA (CDM-BSA) supplemented with 10 ng/mL recombinant Activin A and 12 ng/mL recombinant FGF2 on 0.1% Gelatin and MEF media coated plates. Cells were passaged every 4-6 days with collagenase IV as clumps of 50-100 cells. The culture media was replaced 48 h after the split and then every 24 h. The generation of FUCCI-hESC lines is based on the FUCCI system (Pauklin, S. & Vallier, 2014; Sakaue-Sawano, A. et al. 2008). hESCs were differentiated into endoderm as previously described (Vallier et al, 2009). Following FACS sorting, Early G1 (EG1) cells were collected and immediately placed into the endoderm differentiation media and time-points were collected at 0h, 12h, 24h, 36h, 48h and 72 h. Endoderm specification was performed in CDM with Polyvynilic acid (CDM-PVA) supplemented with 20 ng/mL FGF2, 10 μM Ly-294002 (Promega), 100 ng/mL Activin A, and 10 ng/mL BMP4 (R&D). ChIP has been described earlier in (Pauklin and Vallier 2013; Pauklin et al., 2016; Bertero et al., 2015). All steps were performed on ice or at 4°C and ice-cold buffers and PBS were supplemented with 1mg/ml Leupeptin, 0.2mM PMSF, and 10mM NaButyrate were used unless otherwise stated. Approximately 0.7x10^6 cells were used per sample and cross-linked with 1% formaldehyde for 15 minutes. Cross-linking was stopped by incubating samples with glycine at a final concentration of 0.125M for 5 minutes at room temperature, and the cells were washed with PBS followed by pelleting at 250g for 5 minutes. The pellet was re-suspended in 2ml ChIP Cell Lysis Buffer (CLB: 10 mM Tris pH8, 10 mM NaCl, 0.2% NP-40) and incubated for 10 minutes to lyse the plasma membranes. Nuclei were pelleted at 600g for 5 min, lysed in 1.25ml of ChIP Nuclear Lysis Buffer (NLB: 50 mM Tris pH8, 10mM EDTA, 1% SDS) for 10 minutes, and then 0.75ml of ChIP Dilution Buffer (DB: 20 mM Tris pH8, 2mM EDTA, 150mM NaCl, 0.01% SDS, 1% Triton X-100) was added to the samples. Chromatin was sonicated in 15ml Diagenode Bioruptor Pico water bath sonicator with an automated water-cooling system, by performing 10 cycles of 30 seconds ON, 45 seconds OFF. This protocol resulted in the homogeneous generation of fragments of 100-400bp. Samples were clarified by centrifugation at 16000g for 10 minutes, and diluted with 3.5ml of DB. After pre-clearing with 10µg of non-immune IgG for 1h and 50µl of Protein G-Agarose for 2h, ChIP was performed overnight in rotation using specific antibodies or non-immune IgG as a control. After incubation for 1 hour with 30µl of Protein G-Agarose, beads were washed twice with ChIP Washing Buffer 1 (WB1: 20mM Tris pH8, 2mM EDTA, 50mM NaCl, 0.1% SDS, 1% Triton X-100), once with ChIP Washing Buffer 2 (WB2: 10mM Tris pH8, 1mM EDTA, 0.25M LiCl, 1% NP-40, 1% Deoxycholic acid), and twice with Tris-EDTA (TE: 10mM Tris pH8, 1mM EDTA). Precipitated DNA was eluted with 150µl of ChIP Elution Buffer (EB: 100mM NaHCO3) twice for 15 minutes at room temperature in rotation, and processed as follows in parallel with 300µl of sonicated chromatin non-used for ChIP (Input). Cross-linking was reverted by adding NaCl to a final concentration of 300mM for protein-DNA de-crosslinking and incubated at 65°C for 5 hours and 1µg RNase A (Sigma) to digest contaminating RNA. Finally, 60µg of Proteinase K (Sigma) were added overnight at 45°C. DNA was extracted by sequential phenol-chloroform and chloroform extractions, and precipitated overnight at -80°C in 100mM NaAcetate, 66% ethanol and 50µg of glycogen (Ambion) as a carrier. After centrifugation at 16,000g for 1 hour at 4°C, DNA pellets were washed once with ice-cold 70% ethanol, and finally air dried. Library preparation and sequencing were performed at the Wellcome Sanger Institute next-generation sequencing facility. Size selection was applied, and fragments between 100bp and 400bp (average length of 200bp) were used to prepare barcoded sequencing libraries. 10 ng of input material for each condition were also used for library preparation and later used as a control during peak calling. The libraries were generated by performing 8 PCR cycles for all samples.