|
| Status |
Public on Nov 12, 2015 |
| Title |
Pol2_IP_rrp6_1 |
| Sample type |
SRA |
| |
|
| Source name |
Budding yeast cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
cell density: Log phase (OD= 0.8)
cell cycle stage: Asynchronous
chip antibody: 6ul of Anti-CTD (8WG16 Covance)
|
| Treatment protocol |
Cells were crosslinked with 1% formaldehye at room temperature for 20 min, quenched with glycine and processed for IP
|
| Growth protocol |
Yeast were grown in Yeast Extract Peptone (YEP) media (100ml per IP) with 2% glucose at 30°C
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cell lysis by bead beating (6 cycles of 1 min each) and chromatin fragmentation with 200 units of Mnase at 37˚C. Clarified lysates were IPed with Anti-CTD overnight and followed by incremental salt washes, and phenol chloroform extraction to isolate the IP DNA
Standard BGI protocol was used as follows: i) quality control by Qubit and Agilent 2100 ii) Addition of A base to 3’ end and adapter ligation iii) PCR amplification and size selection for 100-300 bp and, finally iv) Library QC by Agilent 2100 and qPCR.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Description |
Immunoprecipitated DNA
|
| Data processing |
Fastq files were put through the FASTQC program to verify good quality of library
alignment to the sacCer3 genome using Bowtie2 to obtain SAM files. SAMtools was used for SAM to BAM conversion with the FLAGs to discard PCR duplicates and multiple mapping reads.
Each sample was normalized to total library read count (bamCoverage tool in the deepTools package)
After determining strong correlation values, the replicates were summed for further analysis.
IP/Input ratios: Every base of the genome was assigned the total number of sequence reads overlapping it, separately for the input and IP sequence reads. Subsequent normalization and analysis was performed on median read coverage across 100-bp windows, sliding along each chromosome in 50-bp steps. The median IP coverage of each 100-bp interval was divided by the median input coverage for the same window. The IP/input ratios of each interval were normalized, dividing by the median genome- wide IP/input. Positions with fewer than 20 input reads were excluded from all subsequent analysis because of unreliable enrichment associated with division by low numbers.
Genome_build: sacCer3
Supplementary_files_format_and_content: bed files were generated using BEDTools and represent the IP/input ratio in 100bp window, sliding along the chromosome in 50bp steps individually for each mutant, replicates combined. BED files are available on the series record.
|
| |
|
| Submission date |
Sep 03, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Craig L Peterson |
| E-mail(s) |
[email protected]
|
| Phone |
508-856-5858
|
| Organization name |
University of Massachusetts Medical School
|
| Department |
Program in Molecular Medicine
|
| Street address |
373 Plantation Street
|
| City |
Worcester |
| State/province |
MASSACHUSETTS |
| ZIP/Postal code |
01605 |
| Country |
USA |
| |
|
| Platform ID |
GPL13821 |
| Series (2) |
| GSE72692 |
Chromatin dynamics and the RNA exosome function in concert to regulate transcriptional homeostasis (ChIP-seq) |
| GSE73145 |
Chromatin dynamics and the RNA exosome function in concert to regulate transcriptional homeostasis |
|
| Relations |
| BioSample |
SAMN04028212 |
| SRA |
SRX1181998 |
