|
|
GEO help: Mouse over screen elements for information. |
|
Status |
Public on Nov 10, 2010 |
Title |
PolII_total_0H_plus |
Sample type |
SRA |
|
|
Source name |
Macrophages
|
Organism |
Mus musculus |
Characteristics |
strain: C57BL/6 cell type: Bone-marrow-derived macrophages treatment: PolII_total_0H_plus antibody: anti-Pol II total antibody manufacturer: Abcam antibody catalog #: ab5408 antibody lot #: 648628 time: 0H agent #1: GSK-BET
|
Treatment protocol |
For ChIP experiments, BMDMs were pre-incubated with 1 mM of GSK-BET compound in DMSO or DMSO only for 30 minutes. Cells were stimulated with 100 ng/ml LPS for 1 or 4 hours by adding LPS directly to the culture medium.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
For ChIP experiments, BMDMs were pre-incubated with 1 mM of GSK-BET compound in DMSO or DMSO only for 30 minutes. Cells were stimulated with 100 ng/ml LPS for 1 or 4 hours by adding LPS directly to the culture medium. ChIP experiments were performed as described previously 22. Cells were crosslinked with 1% formaldehyde. DNA was sonicated using a Biorupter (Diagenode) to generate DNA-fragments of approximately 200 to 600 bp. The following antibodies were pre-bound to Invitogen Dynal magnetic beads in 0.5% BSA/PBS (Invitrogen Dynabeads anti-mouse M-280 or Dynabeads anti-rabbit M-280): H3 (Abcam, ab1791), H3K4me3 (Millipore, 17-614), H4Acet (Millipore, 06-866), BRD4 (Abcam, ab84776), CDK9 (Santa Cruz, sc-8338x), Pol II (Abcam, ab5408) and Pol II S2 (Abcam, ab5095). Regular ChIP was performed using 1x106 cells (for H3, H3K4me3, Pol II and Pol II S2) or 2x106 cells (for H4Acet, BRD4 and CDK9) and 2 mg antibody coupled to 20 mL beads. ChIP-Seq was performed using 1x107 cells and 7 mg antibody coupled to 70 mL magnetic beads (for H3, H3K4me3 and Pol II) or 2x107 cells and 14 mg antibody coupled to 140 mL beads (for Pol II S2, H4Acet, BRD4 and CDK9). Beads were added to the cell lysates and incubation was allowed to proceed overnight. Beads were washed 8x in modified RIPA wash buffer (50 mM HEPES [pH 7.6], 100 mM LiCl [for H4Acet, BRD4 and CDK9] or 300 mM LiCl [H3, H3K4me3, Pol II and Pol II S2], 1mM EDTA, 1% NP-40 and 0.7% Na-deoxycholate) and 1x in TE containing 50 mM NaCl. Elution of DNA was performed in TE buffer containing 1% SDS. After overnight cross-link reversal at 650C, RNase digestion and Proteinase K digestion, ChIP DNA and input DNA was purified using the QIAGEN Quiaquick PCR purification kit. For regular ChIP and for validation of ChIP-Seq, ChIP DNA was analyzed via qPCR using SYBR Green master mix (Roche) and the LightCycler480 (Roche). For ChIP-Seq, 30 mL of remaining ChIP DNA was used to generate blunt-ended DNA using the Epicenter DNA ENDRepair kit (Epicenter Biotechnologies). The end-repaired DNA was purified using the QIAGEN Quiaquick PCR purification kit. Using Klenow Fragment (NEB) A bases were added to the DNA. DNA was purified using the QIAGEN MinElute kit. T4 DNA ligase (NEB) was used for ligation of Illumina/Solexa adapters to the DNA fragments. The adaptor-ligated DNA was purified with the QIAGEN MinElute kit. The DNA fragments were subjected to 18 cycles of PCR using the Illumina/Solexa primers 1.0 and 2.0 to generate the ChIP-Seq libraries. The ChIP-Seq libraries were purified with the QIAGEN MinElute kit. Samples were sequenced on the Illumina GAIIx platform for 36 cycles, and raw sequencing data was processed using the onboard SCS/RTA software version 2.6 yielding 36bp reads. Sequencing kits used were version 4 sequencing kits.
|
|
|
Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina Genome Analyzer II |
|
|
Description |
Chromatin Immunoprecipitation against Pol II total
|
Data processing |
RTA 2.6, wig files contain alignments to mm9
|
|
|
Submission date |
May 19, 2010 |
Last update date |
May 15, 2019 |
Contact name |
Uwe Schaefer |
E-mail(s) |
uschaefer@rockefeller.edu
|
Phone |
212-327-8265
|
Organization name |
Rockefeller University
|
Lab |
Laboratory of Lymphocyte Signaling
|
Street address |
1230 York Ave.
|
City |
New York |
State/province |
NY |
ZIP/Postal code |
10065 |
Country |
USA |
|
|
Platform ID |
GPL9250 |
Series (1) |
GSE21910 |
Synthetic antagonist of a histone reader reveals and targets genes essential for inflammation |
|
Relations |
SRA |
SRX020990 |
BioSample |
SAMN00013897 |
Named Annotation |
GSM545122_Uwe_PolII_total_0H_plus_100w_20s.wig.gz |
Supplementary file |
Size |
Download |
File type/resource |
GSM545122_Uwe_PolII_total_0H_plus_100w_20s.wig.gz |
22.5 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
Processed data provided as supplementary file |
Raw data are available in SRA |
|
|
|
|
|