Illumina HiSeq 4000 paired end sequencing; ChIP-seq of FOXK2 in human embryonic stem cells H1 ESC, and the differentiated mesendoderm cells and and NPC cells
ChIP-seq of FOXK2 in human embryonic stem cells H1 ESC, and the differentiated mesendoderm cells and and NPC cells
H1 derived NPC FOXK2 rep2_p
ChIP-seq was performed using WiCell H1 Human Embryonic Stem Cells (hESCs) or differentiated Mesendordermal cells (MESE) or neural progenitor cells (NPC). hESCs were grown in mTeSR1 on Matrigel coated culture plates and passaged every 4-6 days with ReleSR as clumps of 50–100 cells. The culture media was replaced daily. hESCs were differentiated into Mesendoderm by culturing in mTeSR1 containing 5 ng/ml BMP4 (R&D systems, 314-BP-010) and 25 ng/ml Activin A (PeproTech 120-14E) for 40 h. hESCs were differentiated into NPC by culturing in STEMdiff™ Neural Induction Medium (Stemcell Technologies) using the monolayer culture method for 7 days with the media changed daily. H1 hESCs cells were grown in mTeSR1 media, MESE cells were grown in mTeSR1 containing 5 ng/ml BMP4 and 25 ng/ml Activin A for 40 h, NPC cells were grown in STEMdiff™ Neural Induction Medium for 7 days . 50 million cell per IP were collected. Approximately 5x10^7 cells were used per sample and cross-linked with 1% formaldehyde for 10 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 10ml ChIP Lysis Buffer I (50 mM HEPES, pH 7.5, 140 mM NaCl, 1 mM EDTA, 10% Glycerol, 0.5% IGEPAL CA-630, 0.25% Triton X-100) and incubated for 10 minutes at 4°C.Nuclei were pelleted at 250g for 5 min, lysed in 10ml of ChIP Lysis Buffer II (200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl, pH 8.0 ) for 10 minutes, and then pelleted and suspended in 3 ml of ChIP Lysis Buffer III (10 mM Tris, pH 8.0, 0.1 M NaCl, 1 mM EDTA, pH 8.0, 0.5 mM EGTA, 0.1% Na Deoxycholate (DOC), 0.5% N-lauroyl sarcosine ). Chromatin was sonicated in Diagenode Bioruptor water bath sonicator with an automated water-cooling system, by performing 15 cycles of 30 seconds ON, 30 seconds OFF. Samples were clarified by centrifugation at 15000g for 10 minutes, and add 1/10 volume 10% Triton X-100. ChIP was performed overnight in rotation using 10 µg FOXK2 antibody (Bethyl Laboratories A301-729A) prebound to 100µl Protein A Dynabeads (Life Technologies Ltd 10002D). Beads were washed five times with ChIP RIPA Washing Buffer (50 mM HEPES, pH 7.5, 1 mM EDTA, 0.7% Na-Deoxycholate (DOC), 1% IGEPAL CA-630, 0.5 M LiCl), and twice with Tris-EDTA (TE: 10mM Tris pH8, 1mM EDTA). Precipitated DNA was eluted with 200µl of ChIP Elution Buffer (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 1%SDS) twice for 30 minutes at 65°C in rotation, and 300µl ChIP elution buffer was added to 100µl of sonicated chromatin as ChIP Input and was processed in parallel with ChIP samples. Cross-linking was reverted by incubation at 65°C overnight and Proteinase K (Merck Life Science, 3115887001 ) 1:100 was added for 1h at 55°C in rotation. DNA was purified using QIAquick PCR purification kit (Qiagen) and eluated into 50 µl H2O. ChIP-seq DNA library was prepared using Illumina TruSeq ChIP Library Prep Kit.