GSM4274460: FOXO1_SUDHL4; Homo sapiens; ChIP-Seq
Nuclei were isolated by resuspending cell pellet in a buffer containing 10mM Tris pH 8.0, 10 mM NaCl, and 0.2% NP-40 and leaving on ice for 30 minutes. Nuclei were pelleted by centrifugation at 7400 rpm, 4C for 30 seconds, and pellet was resuspended in a buffer containing 50 mM Tris pH 8.0, 10 mM EDTA, and 1% SDS and left on ice for 30 minutes with vortexing every 5 minutes. Sample was transferred to a thin-walled 1.5 mL sonication tube and sonicated at low-power setting until chromatin was on average 200-600 basepairs. 250 ug of chromatin was diluted in a 1:4 ratio with IP dilution buffer containing 20 mM Tris pH 8.0, 2 mM EDTA, 150 mM NaCl, 1% Triton X-100, and 0.01% SDS, and precleared with IgG coated protein A+G dynabeads for 3 hours at 4C. The diluted chromatin was then separated from the beads and 5 ug of FOXO1 ChIP-seq grade antibody (abcam) was added to the sample for rotation overnight at 4C. The next morning, protein A+G beads were added and sample was rotated for an additional 3 hours at 4C. Beads were then washed with increasing stringency, then resuspended in DNA elution buffer containing SDS and NaHCO3, and were allowed to shake for 1 hour at 65C. DNA-containing supernatant was removed from the beads into a new tube and DNA crosslinks were reversed overnight at 65C in the presence of NaCl and RNase. Libraries for next-generation sequencing were generated using the NEBNext NGS DNA Library Preparation kit (E7645 and E7500)
GEO Accession
GSM4274460