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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Oct 01, 2013 |
| Title |
POLR3D_Liver_1 |
| Sample type |
SRA |
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| Source name |
Liver
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| Organism |
Mus musculus |
| Characteristics |
cell type: Liver
strain: C57/BL6
age: post natal week 12
chip antibody: anti-POLR3D (mouse and human) CS681 Ab, raised against peptide CSPDFESLLDHKHR {Chong, 2001 #96}
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| Growth protocol |
Cell lines : cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% foetal bovine serum, 4 mM glutamine, 100 units/ml of penicillin, and 100 μg/ml of streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell lines : approximately 10 million subconfluent IMR90 or Hepa 1-6 cells were used per ChIP. The protocol used was similar to the one described by (O'Geen et al. 2006). Cells were directly crosslinked in the culture medium for seven minutes with 1% formaldehyde. The chromatin was sonicated to an average size of 200-600 base pairs.
Mouse livers : livers were perfused with 5 ml of PBS through the spleen, immediately homogenized in PBS containing 1% formaldehyde, and then processed as described in (Ripperger and Schibler 2006).
Aliquots of sonicated chromatin were mixed with different antibodies and incubated overnight at 4˚C on a rotating wheel. Immunoprecipitated material was recovered by addition of 15 µl of protein A agarose beads (pre-blocked with 10 µg/ml of BSA and 10 µg/ml of salmon sperm DNA) and incubation for 1 h at room temperature on a rotating wheel. The beads were washed with dialysis and wash buffer (O’Green et al., 2006). De-crosslinking, RNase A treatment, proteinase K treatment, and DNA purification were performed as described in (O’Green et al., 2006).Ten ng of immunopurified chromatin as well as input DNA was used to prepare sequencing libraries with the ChIP-Seq Sample Preparation Kit (Illumina; San Diego, California, USA; Cat. No IP-102-1001) according to the protocol supplied by the manufacturer
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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 |
Basecalls was performed using Illumina CASAVA pipeline v1.8.2.
Human,respectively Mouse ChIP-seq reads were aligned to the hg18 (resp. mm9) genome assembly via the eland_extended mode of Eland v2e in the Illumina CASSAVA pipeline v1.8.2. The tags with multiple but perfect matches on the genome were then mapped with the “fetchGWI” software.
Tags with up to 500 matches in the genome were attributed a weight corresponding to the number of times they were sequenced divided by the number of matches in the genome.
Scores were calculated as log2((Immunoprecipitation tag counts +30)/(Input tag counts+30)) over regions encompassing the RNA coding sequence as well as 150 base pairs (bp) upstream and 150 bp downstream of the RNA coding region.
Genome_build: hg19 for Human IMR90 cells
Genome_build: mm9 for Mouse liver and hepatocarcinoma cells
Supplementary_files_format_and_content: bedGraph files for genome releases hg18 (human data) and mm9 (Mouse data) showing the regions around Pol3 transcribed features for each IP and for the Inputs.
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| Submission date |
Jun 11, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Nicolo Riggi |
| Organization name |
CHUV
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| Department |
Département de Pathologie Expérimentale
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| Lab |
Institut universitaire de pathologie
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| Street address |
Bugnon 25
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| City |
Lausanne |
| State/province |
VD |
| ZIP/Postal code |
1011 |
| Country |
Switzerland |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE47849 |
POLR3G and POLR3GL-RNA polymerase III target genes |
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| Relations |
| BioSample |
SAMN02199197 |
| SRA |
SRX301451 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1160649_POLR3D_Liver_1.bedgraph.gz |
2.6 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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