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| Status |
Public on May 08, 2018 |
| Title |
JM11_ChIA-PET-2_ZT06 |
| Sample type |
SRA |
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| Source name |
PolII_ChIAPET_single_Xlink
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| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6
genotype/variation: wild type
age: 3-6 months
tissue: Liver
time point: ZT06 (middle of the day)
chip antibody: RNA Polymerase II, clone 8WG16 (Covance, MMS-126R)
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| Growth protocol |
C57BL/6 mice (3-6 month-old) were entrained to a 12 hours light 12 hours dark cycle with food and water provided ad libitum
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Mouse liver were collected at ZT06 (middle of the day) or ZT18 (middle of the night), rinsed in ice-cold 1X PBS and flash-frozen. Frozen livers were crushed to a fine powder in liquid nitrogen using a mortar, and crosslinked with 1% formaldehyde (JM11) or with 1. 5mM EGS and 1% formaldehyde (JM08 and JM12). Nuclei were purified, sonciated, and sonicated. Sonicated chromatin was then used to isolate Pol II -DNA complexes with an antibody.
Nuclei were sonicated in 10 mM Tris pH 7.5, 150 mM NaCl, 2 mM EDTA, 0.5% SDS, 0.2% Triton, 1X protease inhibitor cocktail. Chromatin samples from 3-7 livers were pulled together, diluted 5-fold to obtain a final concentration of ChIP buffer of 10mM Tris-Cl pH 7.5, 150mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS, 1X protease inhibitor cocktail. Pol II ChIP were performed in 50 ml canonical tube with 65 µg of anti-RNA Polymerase II 8WG16 monoclonal antibody (# MMS-126R, Covance). Immunoprecipitated chromatin was washed once with TSE I buffer (10mM Tris pH 7.5, 0.1%SDS, 1% Triton X-100, 2mM EDTA, 150mM NaCl, 1mM DTT, 1 mM PMSF), once with TSE II buffer (10mM Tris pH 7.5, 0.1%SDS, 1% Triton X-100, 2mM EDTA, 500mM NaCl, 1mM DTT, 1mM PMSF), once with LiCl Buffer III (10mM Tris pH 7.5, 0.25M LiCl, 1% NP-40, 1% Na Deoxycholate, 1mM EDTA, 1mM DTT, 1mM PMSF), and once with TET buffer (10mM Tris pH 7.5, 1mM EDTA, 0.1% Triton). Beads were finally resuspended in 2 ml of 1X TE buffer supplemented with 1X TE. Immunoprecipitated chromatin was end-repaired, processed for ligation with biotinylated half-linkers, and 5’ phosphorylated while still complexed with Pol II antibodies on magnetic beads following published protocols. Chromatin was then eluted, diluted to a final volume of 10 ml, and used for the proximity ligation step (performed for a minimum of 16 hours at 4ºC under extremely diluted conditions, i.e., < 0.2 ng DNA/μL, to favor ligation events within individual crosslinked chromatin complexes). Following the proximity ligation step, chromatin was treated with proteinase K, reverse crosslinked, and the DNA purified. ChIA-PET DNA was then digested with MmeI, immobilized on streptavidin beads, and ligated to the ChIA-PET adapters. The DNA samples were finally proceeded through nick translation and a PCR amplification step to generate the library. Libraries were ran on an agarose gel, and fragment of ~229 bp were gel-extracted, purified, and quantified.
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| Library strategy |
ChIA-PET |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina MiSeq |
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| Description |
interaction_genomic_DNA_fragments
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| Data processing |
Reads from the orginal fastq files were processed to extract the tag 1 and 2 (i.e., read 1 and 2) along with their accompanying half-linker code and generate two fastq files (R1 and R2 file) containing the sequence identifier, the raw sequence, and the sequence quality values for each tag.
The R1 and R2 fastq files were aligned to the mouse genome (mm10 version) as paired-end reads using bowtie2 and the options -X 5 and --fast
Paired-end tags with both reads mapping uniquely to the mouse genome were filtered in and all other paired tags removed from the analysis.
Paired-end tags (PETs) were parsed based on the half-linker barcodes into non-chimeric PETs (specific products) or chimeric PETs (non-specific products).
Duplicated PETs were removed for both chimeric and non-chimeric products.
Genome_build: mm10
Supplementary_files_format_and_content: tab-delimitated text file containing all paired-end tags for the 3 biological replicates with the following data columns;
Supplementary_files_format_and_content: Experiment Read1 Tag1 chr start Read2 Tag2 chr start Distance_Read1_Read2
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| Submission date |
Feb 14, 2018 |
| Last update date |
May 08, 2018 |
| Contact name |
Jerome Menet |
| E-mail(s) |
menetlab@gmail.com
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| Organization name |
Texas A&M University
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| Department |
Biology
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| Lab |
Menet Laboratory
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| Street address |
Texas A&M University Biology Department 3258 TAMU College Station, TX 77843
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| City |
College Station |
| State/province |
Texas |
| ZIP/Postal code |
77843 |
| Country |
USA |
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| Platform ID |
GPL16417 |
| Series (2) |
| GSE110603 |
Characterization of tissue-specific BMAL1 cistromes reveals new roles for enhancer-enhancer interactions in regulating rhythmic transcription [ChIA-PET] |
| GSE110604 |
Characterization of tissue-specific BMAL1 cistromes reveals new roles for enhancer-enhancer interactions in regulating rhythmic transcription |
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| Relations |
| BioSample |
SAMN08536203 |
| SRA |
SRX3699481 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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