|
| Status |
Public on Apr 17, 2017 |
| Title |
Pol II ChIP-nexus (1 h 0.5 uM Triptolide treatment) Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Kc167 cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: Kc167 cells
chip antibody: anti-RPB3 rabbit polyclonal, GenScript (custom antibody)
illumina multiplex barcode: GCCAAT
|
| Treatment protocol |
Cells were treated with 0.5 μM Triptolide (TOCRIS Bioscience Cat. No. 3253 dissolved in DMSO) at room temperature for 1 h.
|
| Growth protocol |
grown at 25C in HyClone SFX-Insect Cell Culture Media
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
cells were cross-linked in 1.0% formaldehyde. Cells were then lysed by lysis buffer. Chromatin was sonicated by bioruptor to an average size of 150~500 bp. ~ 10 million cells was used for one ChIP-nexus
DNA-protein-complexes were isolated with specific antibodies when still being attached to protein A-dynabeads. DNA was end repaired using NEBNext End repair module (#E6050L). A single 3'-A overhang was added to the blunted ends using NEBNext dA-Tailing module (#E6053L). Adapters with single 3'-T overhangs and 5’ overhangs (barcode composed of 5 random nucleotides) were ligated to the adenylated fragments. Ligated fragments were blunted again by Klenow 3'-5' exo- polymerase to fill in the 5’ overhang first and then by T4 DNA polymerase to trim any possible 3’ overhang. Blunted DNA was subsequently digested by lambda exonuclease first and RecJf afterwards. Digested single strand DNA was then eluted, reverse cross-linked and purified prior to self-circularization by Circligase. An oligonucleotide was mixed with circularized single DNA for subsequent BamHI digestion to linearize the single DNA again. Linearized single strand DNA was then PCR-amplified using adaptor sequences and library was purified on 2% agarose gel to remove adaptor-adaptor ligation products. Following cluster generation on the flowcell surface, the sequencing libraries were sequenced following Illumina's protocol.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Base calling, read filtering and demultiplexing were performed by Illumina CASAVA 1.8.2 with default settings
Custom barcode sequences were removed from the first 9bp of each read and sequencing adapters were trimmed from the 3' end using cutadapt v1.3
Remaining reads with length >= 22bp were aligned to the reference genome using bowtie v1.0.0, retaining only uniquely aligning reads with a maximum of 2 mismatches
Aligned reads were separated by strand and reduced to a single base at the 5' end
Genome-wide coverage counts were calculated for each strand separately and normalized to reads per million
Replicates are merged together and saved as BigWig format
Genome_build: UCSC dm3
Supplementary_files_format_and_content: BigWig files contain genome coordinates and counts of the first base of aligned reads, separated by alignment strand
|
| |
|
| Submission date |
Mar 06, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Julia Zeitlinger |
| E-mail(s) |
[email protected]
|
| Organization name |
Stowers Institute for Medical Research
|
| Lab |
Zeitlinger lab
|
| Street address |
1000 E 50th Street
|
| City |
Kansas City |
| State/province |
MO |
| ZIP/Postal code |
64110 |
| Country |
USA |
| |
|
| Platform ID |
GPL19132 |
| Series (2) |
| GSE85738 |
Paused RNA polymerase II inhibits new transcriptional initiation [ChIP-nexus] |
| GSE85741 |
Paused RNA polymerase II inhibits new transcriptional initiation |
|
| Relations |
| BioSample |
SAMN06481743 |
| SRA |
SRX2614460 |
