 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Oct 22, 2015 |
| Title |
Abf1_ChEC_seq_40s_(20141219_6) |
| Sample type |
SRA |
| |
|
| Source name |
Abf1 ChEC-seq
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
cell part: nuclei
mutant: WT
strain: W1588-4C (congenic to W303-1A except RAD5+)
genotype: ABF1-3FLAG-MNase-kanMX6
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For each ChEC experiment, a 50 mL culture was grown to an OD600 of 0.5-0.7 at 30C in YPD. Cells were pelleted at 3,000 x g and washed three times with 1 mL Buffer A (15 mM Tris pH 7.5, 80 mM KCl, 0.1 mM EGTA, 0.2 mM spermine, 0.5 mM spermidine, Roche cOmplete EDTA-free mini protease inhibitors, 1 mM PMSF), centrifuging as above between washes. Cells were resuspended in 600 µL Buffer A containing 0.1% digitonin and permeabilized at 30C for 5 min. CaCl2 was added to 2 mM and ChEC digests were performed at 30C. At each time point, a 100 µL aliquot of the digest was transferred to a tube containing 100 µL 2X stop buffer (400 mM NaCl, 20 mM EDTA, 4 mM EGTA) and 1% SDS. Protein was then was digested with 80 µg proteinase K at 55C for 30 m. Nucleic acids were extracted with an equal volume of phenol/chloroform/isoamyl alcohol and precipitated with 2.5 volumes 100% ethanol and 30 µg Glycoblue (Ambion). Pellets were washed once with 1 mL 100% ethanol, dried, and resuspended in 30 µL 0.1X TE buffer, pH 8.0. RNA was digested with 10 µg RNase A at 37C for 20 m. Prior to generating sequencing libraries, ChEC DNA was subjected to size-selection using Ampure XP beads (Agencourt). A beads:sample ratio of 2.5:1 was used and the unbound fraction, containing small DNA fragments, was extracted to remove RNase and precipitated as above, and used for library preparation. Sequencing libraries were constructed as described (PMID 22025700, 24144978), except that KAPA polymerase was used for library amplification.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Abf1 ChEC-seq, 40 second digestion
|
| Data processing |
Library strategy: ChEC-Seq
1. We used Novoalign 2.08.03 (http://www.novocraft.com) to map paired-end 25bp reads to release V64 (Feb 2011) of the S.cervisiae genomic sequence obtained from YeastBase. This release corresponds to release sacCer3 from UCSC . If a read was mapped to multiple locations, one location was picked at random. 2. We extracted properly paired reads (Supplementary file .bed). 3. For each base pair in the genome, we counted the number of paired-end fragment ends aligned to it. 4. We normalized base pair counts by dividing by the total number of fragment ends mapped, and then multiplying by the total number of mapped base pairs (Supplementary file .bedgraph).
|
| |
|
| Submission date |
Mar 31, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Jorja Henikoff |
| E-mail(s) |
jorja@fhcrc.org
|
| Phone |
206-667-4850
|
| Organization name |
Fred Hutchinson Cancer Research Center
|
| Department |
Basic Sciences
|
| Lab |
Henikoff
|
| Street address |
1100 Fairview AV N, A1-162
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109-1024 |
| Country |
USA |
| |
|
| Platform ID |
GPL17342 |
| Series (1) |
| GSE67453 |
ChEC-seq kinetics discriminates transcription factor binding sites by DNA sequence and shape in vivo |
|
| Relations |
| BioSample |
SAMN03453166 |
| SRA |
SRX974335 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1647306.bedgraph.gz |
8.8 Mb |
(ftp)(http) |
BEDGRAPH |
| GSM1647306_Abf1_ChEC_seq_40s.bed.gz |
8.6 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
