|
| Status |
Public on Apr 17, 2017 |
| Title |
Pol II ChIP-nexus with spikein ( (NELF-E and NELF-B knockdown) Rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Kc167 cells
|
| Organism |
Drosophila melanogaster |
| Characteristics |
tissue: Kc167 cells
chip antibody: anti-RPB3 rabbit polyclonal, custom (GenScript)
illumina multiplex barcode: TTAGGC
|
| Treatment protocol |
Cells were treated dsRNA against NELF-B and NELF-E
|
| 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 300~500 bp. ~ 20 million cells was used for one ChIP-nexus
DNA-protein-complexes were isolated with specific antibodies when still being attached to protein A-dynabeads. A fixed amount of spike-in was added to each ChIP in order to control for the loss of Pol II ChIP signal after Triptolide treatment. Human chromatin extracts from a GM12878 cell line were incubated with Dynabeads coupled to antibodies against human Pol II (N20), the entire mixture of Dynabeads and Pol II ChIP was added into each spike-in sample. 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 |
| |
|
| Description |
kc167_nelfe_b_rnai_polii_chipnexus_spikein_normalized_and_merged_positive.bw
kc167_nelfe_b_rnai_polii_chipnexus_spikein_normalized_and_merged_negative.bw
|
| 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 dm3-hg19 combined genome using bowtie v1.0.0, retaining only uniquely aligning reads with a maximum of 2 mismatches
Reads aligned to dm3 genome were separated from reads aligned to hg19 genome
Reads aligned to dm3 genome were separated by strand and reduced to a single base at the 5' end
Genome-wide coverage counts on dm3 genome were calculated and normalized to genome-wide coverage counts on hg19 genome and saved in BigWig format for each strand separately
Genome_build: dm3-hg19 combined genome
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 07, 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) |
| GSE85739 |
Paused RNA polymerase II inhibits new transcriptional initiation [ChIP-nexus spike-in] |
| GSE85741 |
Paused RNA polymerase II inhibits new transcriptional initiation |
|
| Relations |
| BioSample |
SAMN06546416 |
| SRA |
SRX2618949 |
