Illumina HiSeq 2000 paired end sequencing; ChIPseq profile of STAT1 binding after 2h of IFNβ treatment, in bone marrow derived macrophages isolated from WT, Stat1Y701F and Stat1-/- mice.
ChIPseq profile of STAT1 binding after 2h of IFNβ treatment, in bone marrow derived macrophages isolated from WT, Stat1Y701F and Stat1-/- mice.
Bone marrow derived macrophages (BMDM) were obtained by culture of mouse bone marrow in DMEM +10% FCS +10% L-cell-derived conditioned medium containing colony-stimulating factor 1 and antibiotics. The cells were used for the experiments after 10 days in culture. 15x106 cells were seeded on a 15 cm dish and treated with 250 U/mL of mouse recombinant IFNβ for 2 h or left untreated before harvesting. The ChIP was performed using antibody against mouse STAT1 (clone E-23, Santa Cruz). After treatment with IFNβ, formaldehyde was added to the cells to a final concentration of 1% and dishes were incubated for 15 minutes at 37°C. The reaction was quenched by adding 2.5 M glycine to a final concentration of 125 mM for 10 minutes. The fixed cells were washed with PBS and scraped. After centrifugation for 10 minutes at 600g cells were washed with wash I buffer (0.25%Triton X-100; 0.01M EDTA; 0.5 mM EGTA; 0.01 M Hepes) for 10 minutes at 4°C, followed by another centrifugation step. The pellet was resuspended in wash II buffer (0.2M NaCl; 1mM EDTA; 0.5 mM EGTA; 0.01 M Hepes) and kept for 10 minutes at 4°C. Cells were lysed over night with lysis buffer (1% SDS; 0.05M Tris pH 8; 0.01M EDTA) at 4°C. Cells were then sonicated 25 x 30 seconds using a Bioruptor sonicator. This resulted in chromatin fragments with an average length of 300 bp. Samples were centrifuged twice at 16.000g for 15 min and the supernatant was used for the subsequent steps. 1 µL of antibody was added to 25-30 µg of chromatin and rotated overnight at 4°C. Protein G Dynabeads (Invitrogen) were blocked over night in 0.1% BSA and on the next day added to the chromatin/antibody mixture and incubated for 3 hours at 4°C. Beads were then washed for 10 minutes in following buffers consecutively: RIPA buffer (0.15 M NaCl; 0.05 M Tris pH 8; 0.1% SDS; 0.5% NaDoc; 1% NP40), high salt buffer (0.5 M NaCl; 0.05 M Tris pH 8; 0.1% SDS; 1% NP40), LiCl buffer (0.25 M LiCl; 0.05 M Tris pH 8; 0.5% NaDoc; 1% NP40), and two times in TE buffer (1 M Tris pH 8, 0.5 M EDTA). Beads were then shaken in elution buffer (1 mL 10% SDS, 0.5 mL 1M NaHCO3, 50 µL 1M DTT in 5 mL of H2O) at room temperature for 40 minutes and 20 µl 4M NaCl was added to the eluted sample. Samples were incubated at 65°C overnight to reverse the cross-links. Samples were then treated with proteinase K (2 µl ptroteinase K, 8 µl EDTA 0.5 M pH 7.5 and 16 µl 1M TRIS pH 6.5) for 60 min at 55°C. 500 µl Phenol/Chloroform/Isoamyl alcohol (25/24/1, Sigma) was added to all samples and they were then centrifuged for 5 minutes at 16000g. The aqueous phase was used for DNA precipitation. 5-10 ng of DNA precipitate was used for the generation of sequencing libraries using the KAPA library preparation kit for Illumina systems. Libraries were quantified with a Bioanalyzer dsDNA 1000 assay kit (Agilent) and a Q-PCR NGS library quantification kit (KAPA).
Experimental Factor: genotype
Stat1 knock out