|
| Status |
Public on Aug 31, 2012 |
| Title |
Sample 95_RNAPII-Ser5P ChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
RNAPII-Ser5P ChIP-seq
|
| Organism |
Mus musculus |
| Characteristics |
strain background: C57BL/6J
genotype/variation: wild-type
gender: male
tissue: liver
time: CT20
chip antibody: RNAPII-Ser5P
chip antibody vendor: Millipore
chip antibody cat.#: clone3E8.04-1572
chip antibody lot #: NG1881282
|
| Growth protocol |
Male C57BL/6J at 8-12 weeks of age were housed in light-tight boxes and entrained to LD12:12 conditions. After a minimum of 7 days, animals were transferred to constant darkness (DD) conditions. Thirty-six hours after DD, liver samples (N=3 mice) were collected every 4 hrs.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
The immunoprecipitated DNA fragments were repaired by the End-It DNA End Repair Kit (Epicentre Biotechnology) according to the manufacturer’s instructions. The end-repaired ChIP DNA fragments ere purified by MinElute Reaction Cleanup Kit (Qiagen) and eluted in 20 μl in EB buffer.
The resulting DNA fragments were ligated with P1 and P2 adaptors for the Applied Biosystems SOLiD system for 20 min at room temperature using the Quick Ligase Kit (NEB), followed by purification using the MinElute Reaction Cleanup Kit (Qiagen).
The purified, adaptor-ligated ChIP DNA fragments were run on 5% native polyacrylamide gel electrophoresis (PAGE) for an in-gel PCR reaction. A gel slice containing 175–200 bp adaptor-ligated ChIP DNA fragments (corresponding to 115-140 bp genomic gment sizes) was cut and shredded.
PCR Platinum Supermix (100 μl, Invitrogen), 50 pmol of PCR primers with barcodes, 0.5 μl Taq DNA polymerase (NEB), and 0.15 μl p.f.u. Turbo DNA polymerase (Stratagene) were added into the shredded gel slice.
The adaptor-ligated ChIP DNA fragments were amplified by 14-19 cycles of in-gel PCR. After the PCR reaction, gel pieces were filtered out with a 0.45 μm filter spin column, and the amplified ChIP-seq library was purified by the MinElute PCR purification kit (Qiagen).
The library was purified by one more round of 5% PAGE. A gel slice containing 200-230 bp PCR products (110-130 bp fragment size) was cut and shredded, and the amplified library was extracted out of the gel by passive elution in elution buffer (2.5 M ammonium acetate in TE).
Gel pieces were removed with a spin column, and the resulting ChIP-seq library was purified using the QIAquick PCR purification kit (Qiagen).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB 5500xl Genetic Analyzer |
| |
|
| Data processing |
Sequence reads were mapped to the mouse genome (NCBIm37/mm9) with Applied Biosystems BioScope v1.3.
In order to adjust for differences in sequencing depth among time samples, the sequence reads were “down sampled” to the lowest number of the uniquely mapped reads with duplicates among the six time points for each ChIP factor.
PCR duplicates were removed using Picard MarkDuplicates.
Genome_build: mm9
Supplementary_files_format_and_content: The .bw files show the sequence read density across the mm9 mouse genome. The bed files show where the transcription factors or histone marks bind.
|
| |
|
| Submission date |
Aug 08, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Joseph S Takahashi |
| E-mail(s) |
joseph.takahashi@utsouthwestern.edu
|
| Phone |
214-648-1876
|
| Organization name |
University of Texas Southwestern Medical Center
|
| Department |
HHMI, Neuroscience
|
| Lab |
Joseph S. Takahashi
|
| Street address |
5323 Harry Hines Blvd., NA4.118
|
| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390-9111 |
| Country |
USA |
| |
|
| Platform ID |
GPL15907 |
| Series (2) |
| GSE39860 |
Transcriptional Architecture and Chromatin Landscape of the Core Circadian Clock in Mammals |
| GSE39977 |
Transcriptional Architecture and Chromatin Landscape of the Core Circadian Clock in Mammals [ChIP-seq] |
|
| Relations |
| SRA |
SRX174712 |
| BioSample |
SAMN01109414 |
