title: GSM2337199: d4c7 PGCLC, H3K27me3_1; Mus musculus; ChIP-Seq
library_construction_protocol: Cells were suspended in 1 mL of phosphate buffered saline (PBS) and fixed by adding 27 mL of 36.5% formalin (Sigma) for 10 min at room temperature.  Fixation was terminated by adding 110 mL of 1.5 mM glycine.  The fixed cells were washed twice in PBS containing 0.1% bovine serum albmin (BSA), and were lysed in 400 mL SDS-lysis buffer [50 mM Tris-HCl (pH 8.0) containing 1% SDS, 10 uM EDTA, and 1 mM PMSF] on ice for 10 min.  Chromatins were then solubilized using Bioruptor UCD250 with ice-chilled water, with 15 cycles of 30 sec sonication at the high power and 60 sec interval.  The sonication products were centrifuged at 16,000 g for 5 min.  The supernatants were diluted in 1 mL ChIP dilution buffer [16.7 mM Tris-HCl (pH 8.0), 0.01% SDS, 1.1% Triton X100, 1.2 mM EDTA, 167 mM NaCl, 1 mM SDS, 200 mg/mL tRNA (Roche)], and were divided into 0.1 mL as input DNA and 1.3 mL as ChIP samples.  The ChIP samples were mixed with the Dynabeads-antibody complexes and rotated at 4 ºC overnight.  The ChIP-ed Dynabeads were recovered using the DynaMag-2 magnet (Life Technologies), and washed with 200 mL of low salt buffer [20mM Tris-Cl (pH 8.0), containing 0.1% SDS, 1% Triton X100, 2 mM EDTA, and 150 mM NaCl], high salt buffer [20mM Tris-Cl (pH 8.0), containing 0.1% SDS, 1% Triton X100, 2 mM EDTA, and 500 mM NaCl], RIPA buffer [10mM Tris Cl (pH8.0), containing 1% Sodium deoxycholate, 1% NP40, 250 mM LiCl, and 1 mM EDTA], and TE buffer [10 mM Tris Cl (pH8.0), containing 1 mM EDTA].  The ChIP-ed Dynabeads were then incubated in new tubes containing 50 mL of Elution buffer (0.1 mM NaHCO3, 1% SDS, 10 mM DTT, and 0.06 mM tRNA) for 15 min at room temperature.  The supernatants were collected using DynaMag-2, and were reverse-crosslinked by adding 8 mL of 2.5 M NaCl and incubating at 65 ºC overnight.  Then, 2 mL of 0.5 M EDTA and 4 mL of 0.645 mg/mL proteinase K solution were added to reaction mixture, and further incubated at 45 ºC for 1 hr.  The ChIP-ed DNAs were then purified with Qiaquick PCR purification columns, using the buffer EB supplemented with 4 ng/mL tRNA (Qiagen). The ChIP-ed and Input DNAs were sheared to an average size of about 150 bp by ultra-sonication (Covaris, Woburn, MA).  ChIP-ed DNAs obtained from 1×10^6 cells were then subjected to the procedures for SOLiD Fragment Library Preparation (Life Technologies) according to the manufacture’s instruction except for the ligation reaction that was performed overnight.  The libraries were amplified with 11-13 cycles of PCR for H3K4me3 and H3K27ac or 8 cycles for H3K9me2 and H3K27me3.
attributes.tag: GEO Accession
attributes.value: GSM2337199