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| Status |
Public on Jun 28, 2012 |
| Title |
PAX8_Chip-seq |
| Sample type |
SRA |
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| Source name |
PCCL3
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| Organism |
Rattus norvegicus |
| Characteristics |
cell type: Thyroid cells
antibody: PAX8
antibody vendor: Biopat, Milan, Italy
antibody catalog number: PA 0300
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| Treatment protocol |
ChIP samples were prepared from PCCl3 cells as follows: cultures of 10 × 106 cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature. Cross-linking was stopped by the addition of glycine to 125 mM final concentration, and cells were washed twice with 1× PBS. The cell pellet was then resuspended consecutively in different ChIP lysis buffer and sonicated for 90 minutes (30 second pulsing and resting, high frequency) using the Bioruptor sonicator (Diagenode, Denville, NJ) to produce chromatin fragments of 200-500 bp on average. After isolating chromatin, we incubated it with previously prepared PAX8-coated magnetic beads. To prepare beads, 100 ul of magnetic sheep anti-rabbit IgG beads (Invitrogen, Carlsbad, CA) were incubated overnight with 10 ug PAX8 rabbit monoclonal antibody (Biopat, Milan, Italy). The following day, the beads were rinsed and added to shear chromatin and incubated overnight at 4°C. Samples were then rinsed five times with RIPA buffer, and the antibody was stripped from the beads by incubating in 1% SDS at 65°C for 15 minutes; cross-linking was reversed by incubating overnight at 65°C. The next day, samples were sequentially treated with RNAse A and Proteinase K, phenol-chloroform extracted, ethanol precipitated with 20 ug glycogen and resuspended in 50 ul 10 mM Tris pH 8.0.
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| Growth protocol |
PCCl3 thyroid cells were grown in Coon’s modified Ham’s F-12 medium supplemented with 5% donor calf serum and a six-hormone mixture [1 nM TSH, 10 g/ml insulin, 10 ng/ml somatostatin, 5 g/ml transferrin, 10 nM hydrocortisone, and 10 ng/ml glycyl-L-histidyl-L-lysine acetate]
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
IP and non-IP DNAs were modified for sequencing following the ChIP-Seq manufacturer’s protocol (Illumina, San Diego, CA). Briefly, DNAs were blunted with a combination of T4 DNA polymerase, Klenow polymerase, and T4 PNK. Then, a single 3′-end “A” base was added using Klenow exo (3′-to-5′ exo minus). Adapters provided by Illumina were then ligated to the ends of the modified DNA before size selection of ∼200-bp fragments via PAGE extraction. The isolated DNA samples were used as the template for amplification by 18 cycles of PCR, and used for cluster generation on the Illumina 1G genome analyzer. Amplified products were column purified with the QIAquick PCR Purification Kit (Qiagen, Dusseldorf, Germany) and assayed for quantity and quality with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
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| Description |
Chromatin IP against PAX8
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| Data processing |
Alignment: Sequence reads were obtained and mapped to the rat (November, 2004) genome using the MIRO pipeline. All reads mapping with two or fewer mismatches were retained
The reads were directionally extended to 300 bp, and for each base pair in the genome the number of overlapping sequence reads was determined and averaged over a 10 bp window to create a wig file to visualize the data in the University of California Santa Cruz genome browser (http://genome.ucsc.edu)
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| Submission date |
Jan 26, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Pilar Santisteban |
| E-mail(s) |
psantisteban@iib.uam.es
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| Phone |
+44 915854455
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| URL |
http://www.iib.uam.es
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| Organization name |
Biomedical Research Institute-CSIC
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| Department |
Physiopathology of the Endocrine and Nervous System
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| Lab |
2.9_Thyroid Transcripcion
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| Street address |
Arturo Duperier 4
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| City |
Madrid |
| State/province |
Madrid |
| ZIP/Postal code |
28029 |
| Country |
Spain |
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| Platform ID |
GPL10669 |
| Series (2) |
| GSE26871 |
Genome wide profiling of PAX8 binding stes in PCCL3 rat cells |
| GSE26938 |
Global analysis of in vivo Pax8-binding sites in rat thyroid cells using high-throughput technologies: Chip-Seq and whole genome expression arrays |
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| Relations |
| SRA |
SRX039669 |
| BioSample |
SAMN00199226 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM661515_PAX8_PCCL3_IP.bed.gz |
38.0 Mb |
(ftp)(http) |
BED |
| GSM661515_PAX8_PCCL3_IP.wig.gz |
11.9 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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