title: GSM2858421: K562_WT_H3K18Ac_ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: Samples were crosslinked in 1% formaldehyde for 10 minutes at 37°C, washed twice with PBS, and then collected from dishes. For SIRT7, ChIP was carried out with nuclear extracts. First, cells were resuspended in Lysis Buffer 1 (5 mM HEPES, 85 mM KCl, 0.5% NP-40) and incubated for 5 minutes on ice to disrupt cellular membranes. After centrifugation, nuclei were resuspended in Lysis Buffer 2 (1% SDS, 10mM EDTA, 50mM Tris, and protease inhibitors) and incubated on ice for 40 minutes. For H3K18Ac ChIP, whole cell extracts were obtained by directly lysing cells in Lysis Buffer 2 for 40 min on ice. Chromatin was then sonicated to an average fragment size of 150-300bp as analyzed by agarose gel electrophoresis, and subjected to immunoprecipitation with anti-SIRT7 (Cell Signalling, 5360, Lot 1) or anti-H3K18Ac (Abcam, ab1191, Lot GR277130-1) antibodies. ChIPs were washed 5 times with ice cold RIPA buffer (1% NP-40, 0.7% Na deoxycholate, 50mM Tris, 1mM EDTA, 500mM LiCl2, pH 8.0) and crosslinks reversed (1% SDS, 0.1M NaHCO3; 6 hr at 65°C), DNA purified (minElute PCR Purification Kit, Qiagen), and finally the concentration of ChIP and INPUT DNA was measured using PicoGreen (Life Technologies). ChIP libraries were prepared using a ThruPlex DNA-seq library preparation Kit (Rubicon Genomics) following the manufacturer's protocol. ChIP libraries were then size selected with PippinPrep (Sage Science) for a final library fragment size of 350-400bp, and quantified with KAPPA qPCR.
attributes.tag: GEO Accession
attributes.value: GSM2858421