|
| Status |
Public on Apr 05, 2019 |
| Title |
reporter_triptolide_1h_dps_dve_rpb3_chipnexus_rep4 |
| Sample type |
SRA |
| |
|
| Source name |
transfected Kc167 cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: Kc167 cells
treatment: 500 uM Triptolide 1h
chip antibody: anti-RPB3 rabbit polyclonal, GenScript (custom antibody)
|
| Treatment protocol |
Kc167 cells were transfected with a reporter containing promoter sequence of interests. Cells were cultured at 25C for 48 h and treated with Triptolide (500 uM, dissolved in DMSO) or DMSO (2% v/v) at RT for 1h.
|
| Growth protocol |
Kc167 cells were cultured 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 250~500 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. A single 3'-A overhang was added to the blunted ends using NEBNext dA-Tailing module. Adapters with 5’ overhangs were ligated to the adenylated fragments. Libraries were then transformed to blunt end by Klenow 3'-5' exo- polymerase and T4 DNA polymerase. The resulting blunted DNA was digested by lambda exonuclease and RecJf. Digested single strand DNA was then reverse cross-linked and purified. Purified single-stranded DNA was then circularized with CircLigase, annealed with oligonucleotides complementary to the BamHI restriction site and linearized by BamHI digestion. Linearized single strand DNA was then PCR-amplified.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
genome build: dm3-reporter combined genome
Base calling, read filtering and demultiplexing were performed by Illumina CASAVA 1.8.2 with default settings. All samples were first trimmed to 50 bp. Custom barcode sequences were removed from the first 9bp of each ChIP-nexus read or the first 4 bp of each gene-specific 5' RNA sequencing 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 and normalized to reads per million.
Supplementary_files_format_and_content: BigWig files
Supplementary_files_format_and_content: BigWig files contain pileup counts for each base
|
| |
|
| Submission date |
Jan 11, 2019 |
| Last update date |
Apr 07, 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 (1) |
| GSE116244 |
Reporter-ChIP-nexus reveals strong contribution of the Drosophila initiator sequence to RNA polymerase pausing |
|
| Relations |
| BioSample |
SAMN10724526 |
| SRA |
SRX5240984 |
