title: GSM1260395: input_BVSC; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were trypsinized, washed, and collected by centrifugation.  For crosslinking, the pellet was resuspended in PBS containing 1% formaldehyde, incubated for 10 min, and quenched with 125 mM glycine.  The fixed cells were resuspended in 1 ml of cold LB1 (20 mM Tris, 10 mM NaCl, 2.5 mM MgCl2, 0.2% NP-40, and 1 mM PMSF [pH 7.5]), rotated for 10 min at 4 oC, and pelleted by centrifugation at 1,500×g for 5 min.  The pellet was resuspended in 1 ml of LB2 (20 mM Tris, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, and 1 mM PMSF [pH 8.0]) and rotated for 10 min at 4 oC. The nuclei were pelleted by centrifugation at 1,500×g for 5 min at 4 oC and lysed in 400 ml of LB3 (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1mM PMSF).  The lysed nuclei were sonicated using the following conditions: Bioruptor with high power output; 10 cycles (30 s ON and 60 s OFF at 4 oC). Protein-DNA complexes were immunoprecipitated for 15 hrs at 4 oC using 4 μg of antibodies bound to 50 μl of Dynabeads Protein G (Invitrogen).  Immunoprecipitates were sequentially washed with 200 μl of Low salt buffer (0.1 % SDS, 1 % TritonX100, 2 mM EDTA,20 mM Tris, and 150 mM NaCl [pH 8.0]), High salt buffer (0.1 % SDS, 1 % TritonX100, 2 mM EDTA,20 mM Tris, and 500 mM NaCl [pH 8.0]), RIRA buffer (50 mM HEPES-KOH, 0.5 M LiCl, 1 mM EDTA, 0.5% Na-Deoxycholate, and 1% NP-40 [pH 7.4]), and TE buffer (10 mM Tris and 10 mM EDTA [pH 8.0]).  For collection of the protein-DNA complexes, beads were resuspended with 50 μl of elution buffer (100 mM NaHCO3, 1 % SDS, and 10 mM DTT).  The crosslink for both the immunoprecipitated and input DNA fragments was reversed by incubating at 65 oC for 6 hr.  After Proteinase K digestion, the DNA was purified by using PCR purification Kit (Qiagen), and dissolved with TE buffer. DNA of between 2000 and 350 bp, purified using an Agencourt AMPureXP Kit, was sequenced on a Life Technologies SOLiD 5500xl platform to generate single-end 50 bp reads.
attributes.tag: GEO Accession
attributes.value: GSM1260395