title: GSM3677442: KO-NT_mH2A1_ChIP-seq_rep2; Mus musculus; 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 macroH2A1.1 or H3K27me3 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), macroH2A1.1 (Abcam, #ab37264), or anti-H3K27me3 (Cell Signaling C36B11-#9733). 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: GSM3677442