 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Sep 01, 2018 |
| Title |
deltaC4N_b |
| Sample type |
SRA |
| |
|
| Source name |
MATα {leu2-3,112 trp1-1 can1-100 ura3-1 ade2-1 his3-11,15} [phi+], pJL602-PGAL-NLS-brr6∆C4N (LEU2)
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: W303, pPGAL_NLS-brr6deltaC4N
genotype: FLAG-tagged Brr6deltaC4N
molecule: M2 anti-FLAG Affinity purified DNA
|
| Treatment protocol |
Cells (80ml, OD600=1) were crosslinked in 15’ in 1% formaldehyde, quenched 15’ with 125mM glycine
|
| Growth protocol |
6 biological replicates were grown to mid-log phase in YEP media containing 2%Raffinose/0.04% sucrose, diluted into media containing 2% galactose/0.04% sucrose and grown O/N.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were washed 2x in TBS, and resuspended in IP buffer (50 mM Tris [pH 7.4], 125 mM KCl, and 0.1% NP40) plus protease inhibitors (100 μM phenylmethanesulphonylfluoride [PMSF], Sigma and Complete protease tablet (Roche). Cells lysates generated by bead beating (5x1', separated by 2' on ice) were bath-sonicated 4x 7.5’ and clarified 2x 10' at 18,000 RCF. Extracts were incubated (350ul/sample) O/N with 40ul EZview Red anti-FLAG M2 affinity gel (Sigma) and washed twice with 1ml lysis buffer, once with 1ml lysis buffer + 500mM NaCl at 4˚C and once with TE at room temperature.
Crosslinks were reversed and DNA libraries prepared as described in Inada, M. et al.,(2016). NAR 44(19):9180-9189 (PMID:27402158 except that the Thermo Fast End Repair Kit was used for DNA end repair.
Libraries were size selected (200-400bp) and quality checked by bioanalyzer using the Agilent High Sensitivity DNA Kit prior to multiplexing for Illumina sequencing (single end, 50bp reads).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Illumina adaptor removed from read 3' ends using Cutadapt
Reads were aligned to the S288C genome with bowtie v 1.1.2
To identify regions of Brr6 binding, smoothed ChIP signal from the tagged samples (B6N) were divided by the signal from the untagged sample (PJL) after normalizing to the total number of aligned reads. A sliding window of 200 bp moving in increments of 20 bp was used to detect regions that had at least 2-fold enrichment of signal in the tagged sample over the untagged sample. Regions that had at least 50% overlap in both replicates were selected. Only the overlapping portion of the region was considered for further analysis steps. Finally, the average read density (in RPKM) was calculated in each region of Brr6 binding for tagged and untagged and then normalized to the signal from the corresponding whole cell extract from that sample.
Genome_build: R64-2-1_20150113
|
| |
|
| Submission date |
Apr 27, 2018 |
| Last update date |
Sep 01, 2018 |
| Contact name |
Anne de Bruyn Kops |
| E-mail(s) |
annedebk@gmail.com
|
| Phone |
4154250116
|
| Organization name |
UCSF
|
| Department |
Biochemistry and Biophysics
|
| Lab |
Christine Guthrie
|
| Street address |
UCSF Mission Bay, Genentech Hall N376, Guthrie Lab, 600 16th St
|
| City |
San Francisco |
| State/province |
California |
| ZIP/Postal code |
94158 |
| Country |
USA |
| |
|
| Platform ID |
GPL21656 |
| Series (1) |
| GSE113746 |
Brr6 Plays a Role in Gene Recruitment and Transcriptional Regulation at the Nuclear envelope |
|
| Relations |
| BioSample |
SAMN08993117 |
| SRA |
SRX4003432 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3118772_deltaC4N_b_S21_R1_001_multi_sorted.bedgraph.gz |
56.7 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
