 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Dec 01, 2021 |
| Title |
191113L05_MS537c1_3xFLAG-WHI5_WIQ-ChIP |
| Sample type |
SRA |
| |
|
| Source name |
cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: MS537c1
chip-antibody: anti-FLAG antibody (M2, SIGMA Cat# F1804)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde (30 minutes), quenched with 0.125 M glycine (5 minutes), washed twice in cold PBS, pelleted, snap-frozen and stored at −80°C. Pellets were lysed in 300 µL FA lysis buffer (50 mM HEPES–KOH pH 8.0, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM PMSF, Roche protease inhibitor) with ∼1 mL ceramic beads using a Fastprep-24 (MP Biomedicals). The lysate was then collected and sonication to ~200bp fragments. Cell debris was then pelleted and the supernatant retained for ChIP. For each ChIP reaction, 30 µL Protein G Dynabeads (Invitrogen) were blocked (PBS + 0.5% BSA), prebound with 10 µL anti-V5 antibody (SV5-Pk1, BioRad Cat# MCA1360G) or 10 µL anti-FLAG antibody (M2, SIGMA Cat# F1804) and washed once with PBS before incubation with supernatant (4°C, overnight). Dynabeads were then washed (5 minutes per wash) twice in FA lysis buffer, twice in high-salt (500 mM NaCl) FA lysis buffer, twice in ChIP wash buffer (10 mM TrisHCl pH 7.5, 0.25 M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA, 1 mM PMSF) and once in TE wash buffer (10 mM TrisHCl pH 7.5, 1 mM EDTA, 50 mM NaCl). DNA was eluted in ChIP elution buffer (50 mM TrisHCl pH 7.5, 10 mM EDTA, 1% SDS) at 65°C for 15-20 minutes. Eluted DNA was incubated to reverse crosslinks (65°C, 5 hours), before treatment with RNAse A (37°C, 1 hour) and then Proteinase K (65°C, 2 hours). DNA was purified using the ChIP DNA Clean & Concentrator kit (Zymo Research).
Indexed sequencing libraries were prepared using the NEBNext Ultra II DNA Library Prep kit (NEB, # E7645) and pooled before paired-end (2x150 bp) Illumina sequencing
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
191113L05_MS537c1_3xFLAG-WHI5_WIQ-ChIP
|
| Data processing |
read alignment, ChIP-seq Bowtie 1.0.1
read alignment, RNA-seq STAR-2.5.3a
custom code
Genome_build: custom
Supplementary_files_format_and_content: bedGraph of read coverage, normalized to RPM
|
| |
|
| Submission date |
Feb 26, 2021 |
| Last update date |
Dec 01, 2021 |
| Contact name |
Georgi Kolev Marinov |
| Organization name |
STANFORD UNIVERSITY
|
| Department |
Genetics
|
| Street address |
279 Campus Drive West, Beckman Center, B-257A/259
|
| City |
Stanford |
| State/province |
California |
| ZIP/Postal code |
94305-5101 |
| Country |
USA |
| |
|
| Platform ID |
GPL27812 |
| Series (1) |
| GSE167842 |
Transcriptional and chromatin-based partitioning mechanisms uncouple protein scaling from cell size |
|
| Relations |
| BioSample |
SAMN18079093 |
| SRA |
SRX10182757 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5113895_191113L05_MS537c1_3xFLAG-WHI5_WIQ-ChIP.bigWig |
44.4 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
