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| Status |
Public on Jul 06, 2022 |
| Title |
CHIP-seq with M2 mouse anti-Flag antibody |
| Sample type |
SRA |
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| Source name |
Embyonic
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| Organism |
Mus musculus |
| Characteristics |
tissue: Embyonic
cell line: 3T3
cell type: Fibroblast
genotype: CCND1-/- Flag-CCND1
chip antibody: Monoclonal ANTI-FLAG M2, Sigma-Aldrich
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| Treatment protocol |
No treatment
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| Growth protocol |
Cyclin D1 knock-out Mouse embryonic fibroblasts (Ccnd1-/- MEFs) stable transfected with flag-cyclin D1 were maintained in Dulbecco's modified Eagle's media (DMEM). supplemented with 10% FBS, 2 mmol/L glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin in humidified atmosphere containing 5% CO2 at 37°C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cyclin D1-/- 3T3s transduced with MSCV-FLAG/Cyclin D1 cells were fixed with 1% formaldehyde for 15 min and quenched with 0.125 M glycine. Chromatin was isolated by adding 5-10 ml lysis buffer containing PIPES, Igepal, PMSF and Protease Inhibitor Cocktail, followed by disruption with a Dounce homogenizer. Samples were pelleted by centrifugation and resuspended in buffer containing Na deoxycholate, SDS, and Triton X-100. Lysates were sonicated using a Misonix Sonicator 3000 equipped with a microtip in order to shear the DNA to an average length of 300-500 bp. Lysates were cleared by centrifugation and the chromatin suspensions were transferred to new tubes and stored at -80 C. Prior to use in ChIP, protein G agarose beads (Invitrogen) were preblocked using blocking proteins and nucleic acids for 3 hr. For each ChIP reaction, an aliquot of chromatin (20-30 ug) was precleared with 30 ul preblocked protein G agarose beads for 1-2 hr. ChIP reactions were set up using precleared chromatin and antibody (mouse IgG, anti-FLAG M2) in a buffer containing Na deoxycholate and incubated overnight at 4 C. Preblocked protein G agarose beads were added and incubation at 4 C was continued for another 3 hr. Agarose beads containing the immune complexes were washed two times each with a series of buffers consisting of the deoxycholate sonication buffer, high salt buffer, LiCl buffer, and TE buffer. An SDS-containing buffer was added to elute the immune complexes from the beads, and the eluents were subjected to RNase treatment at 37 C for 20 min and proteinase K treatment at 37 C for 3 hr. Crosslinks were reversed by incubation overnight at 65 C, and ChIP DNAs were purified by phenol-chloroform extraction and ethanol precipitation. ChIP DNA was amplified using the Illumina ChIPSeq DNA Sample Prep Kit. In brief, DNA ends were polished and 5’-phosphorylated using T4 DNA polymerase, Klenow polymerase and T4 polynucleotide kinase. After addition of 3’-A to the ends using Klenow fragment (3’-5’ exo minus), Illumina genomic adapters were ligated and the sample was size-fractionated (~250 bp) on a 2% agarose gel. After a final PCR amplification step (18 cycles, Phusion polymerase), the resulting DNA libraries were quantified and tested by QPCR at the same specific genomic regions as the original ChIP DNA to assess quality of the amplification reactions.
DNA libraries were sequenced on a Genome Analyzer II. Sequences (36 nt reads) were aligned to the mouse genome (NCBI Build 37.1/mm9) using Eland (Illumina pipeline) software. Aligned sequence fragments were extended in silico at their 3’-ends to a length of 160 bp, which is the average genomic fragment length in the size selected library, and assigned to 32-nt bins along the genome. The resulting histograms were stored in BAR (Binary Analysis Results) files.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
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| Data processing |
Peak locations were determined using the MACS algorithm. These files were analyzed using Genpathway proprietary software that provides comprehensive information on genomic annotation, peak metrics and sample comparisons for all peaks (intervals). The MACs algorithm comparing cyclin D1/FLAG with IgG (cut off p=10e-5); 3231 peaks were identified, 9 of which had a corresponding peak in the IgG sample (using a threshold of fragment density >8).
To view genomic peaks the BED files were uploaded to the Integrated Genome Browser (Affymetrix) to generate and visualize genomics regions bound by cyclin D1.
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| Submission date |
Jul 01, 2022 |
| Last update date |
Jul 06, 2022 |
| Contact name |
Xuanmao Jiao |
| E-mail(s) |
xuanmao.jiao@gmail.com
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| Organization name |
Baruch S. Blumberg Institute
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| Department |
Pennsylvania Cancer and Regenerative Medicine Research Center
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| Street address |
100 E Lancaster Ave, LIMR R234
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| City |
Wynnewood |
| State/province |
PA |
| ZIP/Postal code |
19096 |
| Country |
USA |
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| Platform ID |
GPL9250 |
| Series (1) |
| GSE207361 |
Binding of cyclin D1 with genomic DNA revealed by CHIP-seq in mouse embryonic fibroblasts (MEFs) |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM6284974_2_Flag_s_2_export_dedup_hits.bed.gz |
124.6 Mb |
(ftp)(http) |
BED |
| GSM6284974_2_Flag_s_2_export_dedup_signal.bar.gz |
90.4 Mb |
(ftp)(http) |
BAR |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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