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Sample GSM545103 Query DataSets for GSM545103
Status Public on Nov 10, 2010
Title 1_CDK_0H_minus
Sample type SRA
 
Source name Macrophages
Organism Mus musculus
Characteristics strain: C57BL/6
cell type: Bone-marrow-derived macrophages
treatment: 1_CDK_0H_minus
antibody: anti-CDK9 (P-TEFb)
antibody manufacturer: Santa Cruz
antibody catalog #: sc-8338x
antibody lot #: unknown
time: 0H
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 CDK9 (P-TEFb)
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 SRX020971
BioSample SAMN00013878
Named Annotation GSM545103_Uwe_1_CDK9_0H_minus_fastq_prefilter_bowtie.sam.bed.wig.gz

Supplementary file Size Download File type/resource
GSM545103_Uwe_1_CDK9_0H_minus_fastq_prefilter_bowtie.sam.bed.wig.gz 28.6 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Processed data provided as supplementary file
Raw data are available in SRA

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