GSM1314682: 20091006_1881_X_BMM_LPS_0120_P65; Mus musculus; ChIP-Seq
For ChIP–Seq analysis, formaldehyde-fixed cells were sonicated and processed for immunoprecipitation. In brief, 3 × 107 BMMs were fixed for 10 min in 1% formaldehyde in PBS, washed in PBS, and harvested by scraping in PBS. Cells were lysed by resuspending cell pellets in RIPA buffer (10mM Tris-HCl, pH8.0, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, 1% Deoxcholic acid sodium salt) and drawing the cell suspensions three times through a 30 gauge needle.  Chromatin was sheared using a probe sonicator (130 W Ultrasonic Processor with a 3mm tip, 5 × 60s at 30% maximum setting). Sonication quality was checked by gel electrophoresis.  Protein concentrations in the extracts were determined (BioRad DC Protein Assay Kit I # 500-111) and sonicated cell extracts containing 0.5 mg protein were incubated with antibodies overnight at 4oC. Immune complexes were recovered by incubation with a 50%-50% mix of magnetic beads coated with Protein A or Protein G (Dynabeads Protein A Invitrogen # 100.02D, Dynabeads Protein G Invitrogen # 100.04D). The magnetic beads were washed with RIPA buffer and the chromatin was eluted with 1% SDS in TE buffer (10 mM Tris-HCl pH8.0, 1 mM EDTA pH8.0) at 65oC for 15 min. Eluted chromatin was reverse-cross-linked by adding 226 mM NaCl and incubating at 65oC for > 5 hr. Then 36 mM Tris–HCl pH8.0, 9 mM EDTA, and 1.5 U of Proteinase K (Fermentas # EO0491) was added to the samples and they were incubated at 42oC for > 1 hr.  DNA was purified using phenol/chloroform/isoamyl alcohol (25:24:1, v:v:v) extraction. The purified immunoprecipitated DNA was prepared for sequencing with the Illumina ChIP-Seq Sample Prep Kit and processed according to the manufacturer’s instructions (Illumina Part # 11257047 Rev. A). A sequencing library for the Illumina Genome Analyzer was derived from the IP using the Illumina reagent kit (see systemsimmunology.org). Single-ended, 36-cycle sequencing was performed on an Illumina Genome Analyzer, and the raw image data were processed using the Illumina Genome Analysis Pipeline Software on a dedicated sequence data processing system (see Genome Analyzer Pipeline Software User Guide, Illumina, San Diego, CA, USA, v0.3).
GEO Accession
GSM1314682