title: GSM2305572: Input_ChIP; Mus musculus; ChIP-Seq
library_construction_protocol: Approximately 2 x 10^7 cells were harvested and crosslinked at room temperature with 1% formaldehyde for 9 minutes. Crosslinking reaction was quenched with the addition of glycine to a final concentration of 125 mM for at least 15 minutes. Crosslinked cells were lysed on ice using 1 ml low-salt ChIP buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.5% NP-40, 1.0%Triton X-100) and sonicated using a Qsonica Sonicator Q500 (Newtown, CT 0647) with the following settings: 70% amplitude; 10 seconds on/20 seconds off cycles; total sonication time of 15 minutes to yield fragments of 500-100 bp. Sonicated samples were centrifuged and the soluble chromatin (supernatant) was used for chromatin immunoprecipiation reactions. Briefly, 200 µl of lysate (~ 2 x 106 cells) was incubated with each antibody, 10µg / IP of HIF1A antibody #AF1935 Immunoprecipitated chromatin was recovered using recombinant protein G beads or magnetic Dynabeads® (Invitrogen). ChIP samples were reverse crosslinked and DNA was purified using the phenol-chloroform extraction method. Illumina ChIP Seq libraries were prepared as follows: ChIP-isolated DNA was size selected by 2% Agarose Nusieve gel, and a 200-500bp region was excised and purified using Qiaquick gel extraction kit. Adapter linker attachment was performed by PCR using 30ng of DNA and 2X Phusion master mix and primers, using conditions of 30 sec, 98°C, followed by 10 sec, 98°C, 30 sec, 65°C and 30 sec, 72°C with cycle number empirically determined, followed by a final 5 min, 72°C extension step. Adapter ligated PCR reactions were purified using Agencourt Ampure XP PCR Purification Beads per manufacturer’s protocol.
attributes.tag: GEO Accession
attributes.value: GSM2305572