|
| Status |
Public on May 09, 2023 |
| Title |
5min_V5Rap1 ChIP rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Saccharomyces cerevisiae + paradoxus
|
| Organisms |
Saccharomyces cerevisiae; Saccharomyces paradoxus |
| Characteristics |
tissue: Saccharomyces cerevisiae + paradoxus
cell line_(strain_-_cerevisiae): JRy15237
cell line_(strain_-_paradoxus): JRy15212
genotype (cerevisiae): can1-100, his3-11,15, leu2-3,112, lys2, trp1-1, ura3, fpr1::NAT, RPL13A-2xFKBP12::TRP1 Rap1(134)-FRB_2xV5 bar1delta::k.l.URA3 hmlalphadelta::UniqueHMLwithSNPs TOR1(S1972I); hmrdelta::HygMX
genotype (paradoxus): Z1.1 ho::HygMX ura3::KanMXBarcode TRP LEU HIS Rap1-2xV5
treatment (growth): 5min treatment with 7.5uM Rapamycin
treatment (antibody): 50uL DynaBeads Protein G (ThermoFisher Scientific 10003D) magnetic beads coupled with 5uL monoclonal mouse-anti-V5 antibody (ThermoFisher Scientific R96025)
|
| Treatment protocol |
Additions of rapamycin (at a final concentratiion of 7.5uM) were staggered such that all time points were ready at the same OD (~0.8). ~5×108cells were collected for each sample.
|
| Growth protocol |
Yeast were cultured at 30° in YPD. Cells were grown overnight in YPD, then back-diluted to OD600 ~ 0.1 in 50mL YPD the following day and grown for two-three doublings and then collected at OD600 ~ 0.8.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were lysed by mechanical disruption in a bead beater. Chromatin was fragmented by sonication. All immunoprecipitations were performed using 50uL DynaBeads Protein G (ThermoFisher Scientific 10003D) magnetic beads coupled with 5uL monoclonal mouse-anti-V5 antibody (ThermoFisher Scientific R96025) DNA was purified using Qiagen spin column kit
NEB Ultra II library prep kit
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
FASTQ generation using Illumina Casava v2.2.
Raw reads were mapped to a hybrid SacCer3 genome modified to include "UniqueHML" which has synonymos SNPs at HML to distinguish from MAT as well as hmrdelta::Hyg and the S. paradoxus CBS432 genome assembly, using Bowtie2 with following options: --local --soft-clipped-unmapped-tlen --no-unal --no-mixed --no-discordant
PCR duplicates were removed using, sequentially, SAMtools -fixmate -sort -markdup
bam files were generated using samtools -view -sort -index
samples (121-124,141-144) were also used to generate bedgraphs to compare Rap1 enrichment at MAT in SIR vs sir4delta cells (figure S2H). In these files, bedgraph files were generated that included S. cerevisiae reads only between 0-500bp (average fragment size from sonication) and normalized to the S.cerevisiae genome-wide median (median excluded subtelomeres, chromosome III, and rDNA).
Assembly: SacCer3 (modified)
Supplementary files format and content: bedgraph (normalized to genome-wide median)
|
| |
|
| Submission date |
Mar 20, 2023 |
| Last update date |
May 09, 2023 |
| Contact name |
Eliana Rose Bondra |
| Organization name |
University of California, Berkeley
|
| Department |
Molecular and Cell Biology
|
| Lab |
Rine
|
| Street address |
440 Barker Hall
|
| City |
Berkeley |
| State/province |
California |
| ZIP/Postal code |
94720 |
| Country |
USA |
| |
|
| Platform ID |
GPL33270 |
| Series (2) |
| GSE227761 |
Context dependent function of transcriptional regulator Rap1 in gene silencing and activation [ChIP-seq timecourse] |
| GSE227763 |
Context dependent function of transcriptional regulator Rap1 in gene silencing and activation |
|
| Relations |
| BioSample |
SAMN33830862 |
| SRA |
SRX19731402 |
