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Status |
Public on Dec 01, 2021 |
Title |
191113L02_MS535_LacI-GFP-NLS-5xFLAG-ChIP |
Sample type |
SRA |
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Source name |
cells
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Organism |
Saccharomyces cerevisiae |
Characteristics |
strain: MS535 chip-antibody: anti-FLAG antibody (M2, SIGMA Cat# F1804)
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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
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
191113L02_MS535_LacI-GFP-NLS-5xFLAG-ChIP
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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
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Submission date |
Feb 26, 2021 |
Last update date |
Dec 01, 2021 |
Contact name |
Georgi Kolev Marinov |
Organization name |
STANFORD UNIVERSITY
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Department |
Genetics
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Street address |
279 Campus Drive West, Beckman Center, B-257A/259
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City |
Stanford |
State/province |
California |
ZIP/Postal code |
94305-5101 |
Country |
USA |
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Platform ID |
GPL27812 |
Series (1) |
GSE167842 |
Transcriptional and chromatin-based partitioning mechanisms uncouple protein scaling from cell size |
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Relations |
BioSample |
SAMN18079096 |
SRA |
SRX10182754 |
Supplementary file |
Size |
Download |
File type/resource |
GSM5113892_191113L02_MS535_LacI-GFP-NLS-5xFLAG-ChIP.bigWig |
46.5 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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