GSM5525256: SOX17 ChIP hPGCLC rep2; Homo sapiens; ChIP-Seq
Pellets were thawed on ice and resuspending in lysis buffer at room temperature, rotating for 15 minutes. The resulting lysate was pelleted by 5 minutes of centrifugation at 4000 RPM. Pellet was re-suspended in Nuclei isolation buffer at 4°C, rotating for 10 minutes followed by 5 minutes of centrifugation at 4000 RPM. The resulting pellet was resuspended in 10 mM Tris HCl pH 8, 10 mM EDTA, 0.5 mM EGTA with protease inhibitors. Samples were sonicated and insoluble material was removed. 10% of resulting soluble supernatant was saved as an input sample. To pre-clear, Protein A beads were washed in dilution buffer three times. Protein A beads were resuspended in dilution buffer and added to samples so that Protein A Dynabeads were suspended in a volume of dilution buffer equal to the volume of soluble material. Chromatin was pre-cleared by incubation with Protein A Dynabeads for 2 hours, rotating at 4°C. Beads were removed and anti-SOX17 antibody (AF1924, R and D Systems) or anti-NANOG antibody (AF1997, R and D Systems) were added and allowed to incubate rotating at 4°C overnight. Antibodies were bound for recovery using Protein G Dynabeads by incubation at 4°C, rotating for two hours. Antibody-bound beads were washed and chromatin was eluted from beads by incubating at 65°C, rotating at 1400 RPM in 50mM Tris HCl pH8, 1mM EDTA, 1% SDS two times. To facilitate crosslinking reversal, eluate was left to incubate at 65°C overnight. Eluate was treated with RNase A at 37°C for 30 minutes followed by treatment with Proteinase K at 56°C. DNA was purified using Qiagen PCR purification kit according to manufacturer's instructions. ChIP-seq libraries were generated using using Tecan Genomics Ovation UltraLow V2 DNA-seq (0344NB, Redwood City, CA) according to the manufacturer's instructions. All ChIP-seq libraries were sequenced using a NovaSeq 6000 (Illumina, San Diego) on an NovaSeq SP lane using paired-end 100 base pair reads.