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| Status |
Public on Feb 04, 2015 |
| Title |
Thio_SREBP1_GW3965_6h |
| Sample type |
SRA |
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| Source name |
Primary thioglycollate-elicited peritoneal macrophages
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| Organism |
Mus musculus |
| Characteristics |
strain: C57Bl6
chip target: SREBP-1
chip antibody: sc-13551, sc-8984, sc-367
genotype: wild-type
treatment: GW3965 6h
cell type: macrophages
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| Treatment protocol |
Cells were cultured with no treatment overnight. The next day, cells were treated with GW3965 (1uM) for another 6 h.
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| Growth protocol |
Primary cells were isolated from 6-8 week-old C57Bl/6 mice. Thioglycollate-elicited macrophages were isolated by peritoneal lavage 3-4 days following peritoneal injection of 2.5 ml thioglycollate. Cells were plated in RPMI medium 1640 and 10% fetal bovine serum, washed after adherence and again fed with fresh medium.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
20-40 million macrophages were used per immunoprecipitation. Cells were first crosslinked in 2mM disuccinimidyl glutarate (Thermo Fisher Scientific, Rockford, IL, USA) in PBS for 30 minutes, then subsequently in 1% formaldehyde (Sigma-Aldrich, St. Louis, MO, USA) in PBS for 15 minutes all at room temperature. The reactions were quenched by adding glycine (Sigma-Aldrich, St. Louis, MO, USA) to a final concentration of 125 mM, and the cells were centrifuged and washed twice with ice-cold PBS and swollen for 10 min on ice in hypotonic buffer. Nuclei were then pelleted and resuspended in RIPA lysis buffer (10mM Tris/HCl, 1mM EDTA, 1mM EGTA, 0.1% SDS, 0.5% Na-Deoxycholate, 1% NP-40, 150mM NaCl). Chromatin was sheared to an average DNA size of 100-400 bp by administering 10 pulses of 30 seconds duration at 12 W power output with 1 min pause on wet ice using a Misonix 3000 sonicator. Extracts were clarified by centrifugation at 14,000 rpm for 15 min at 4ºC, pre-cleared with 100 μl of CL4B sepharose beads (Sigma-Aldrich, St. Louis, MO, USA) for 1 hr at 4ºC, then incubated overnight with appropriate specific antibody on rotator. Protein-DNA complexes bound to antibodies were collected by incubation with 40 μl of Protein A beads (GE Healthcare, Pittsburgh, PA, USA) for 2 hr at 4ºC on rotator. Beads were washed 3 times with RIPA lysis buffer, 6 times with LiCl buffer (100mM Tris/HCl, 500mM LiCl, 1% NP-40, 1% Na-Deoxycholate, 1mM EDTA) and twice with TE. Immunoprecipitated chromatin was eluted twice with 100 μl elution buffer (0.1M NaHCO3, 1% SDS) followed by overnight incubation at 65ºC adding NaCl. Samples were treated with RNase A and proteinase K. DNA was isolated using the ChIP DNA Clean & Concentrator kit (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instruction.
Libraries were prepared according to Illumina's instructions. Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (minus exo) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation, ChIP DNA was PCR-amplified with Illumina Genomic adaptors or with NEXTflex DNA barcodes adaptors for 18 cycles and library fragments (ChIP) (insert plus adaptor and PCR primer sequences) were size-selected (150-250bp) from a 2% agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on Illumina Genome Analyzer II or Illumina HiSeq 2000 according to the manuactuere’s instructions.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Libraries were sequenced 50 cycles on an Illumina HiSeq 2000 according to the manufacturer’s instructions. Reads were aligned to the mm9 genome (NCBI build using bowtie (v0.12.7), keeping only reads that mapped best to a single, unique location. Aligned read files were analyzed with HOMER (http://biowhat.ucsd.edu/homer/) to find peaks. Each ChIP-Seq experiment was normalized to a total of 10^7 uniquely mapped tags by adjusting the number of tags at each position in the genome to the correct fractional amount given the total tags mapped.
Genome_build: mm9
Supplementary_files_format_and_content: UCSC Genome Browser BigWig text files were created by calculating ChIP-Fragment pileups given a fragment size of ~150 bp.
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| Submission date |
Nov 28, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Daniel Alameda |
| E-mail(s) |
daniel.alameda@gmail.com
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| Organization name |
CIMA
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| Street address |
Av. de Pío XII, 55
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| City |
Pamplona |
| ZIP/Postal code |
31008 |
| Country |
Spain |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE63697 |
Retinoid X receptors orchestrate osteoclast differentiation and postnatal bone remodeling [SREBP] |
| GSE63698 |
Retinoid X receptors orchestrate osteoclast differentiation and postnatal bone remodeling |
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| Relations |
| BioSample |
SAMN03225191 |
| SRA |
SRX769795 |
| Named Annotation |
GSM1555715_Sample_Thio_SREBP1_GW3965_6h.ucsc.bigWig |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1555715_Sample_Thio_SREBP1_GW3965_6h.ucsc.bigWig |
24.8 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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