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| Status |
Public on Nov 01, 2023 |
| Title |
211117L28_exp211021_MS67_SCGE-T04_anti-Rpb1 |
| Sample type |
SRA |
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| Source name |
211117L28_exp211021_MS67_SCGE-T04_anti-Rpb1
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| Organisms |
Saccharomyces cerevisiae; Nakaseomyces glabratus |
| Characteristics |
sample: Eluted Rpb1 ChIP DNA from a mixture S. cerevisiae (MS67) and C. glabrata (MS99). MS67 was arrested in G1 and collected at timepoint 4.
cell type: yeast
chip-antibody: anti-Rpb1 (8wG16; Millipore # 05-952-I)
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| Growth protocol |
S. pombe: Standard yeast cell culture practices were followed. Cells were cultured at 30C in Edinburgh Minimal Media (EMM).
C. Glabrata: Standard yeast cell culture practices were followed. Cells were cultured at 30C in synthetic complete media with either 2% glucose (SCD) or 2% glycerol + 1% ethanol (SCGE) as a carbon source.
S. cerevisiae: Standard yeast cell culture practices were followed. Cells were cultured at 30C in synthetic complete media with either 2% glucose (SCD) or 2% glycerol + 1% ethanol (SCGE) as a carbon source.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
S. cerevisiae cultures were combined with spike-in cultures (C. glabrata or S. pombe) and immediately fixed with formaldehyde at a final concentration of 1% for 15 minutes. Cells were then quenched with 0.125 M glycine (5 minutes), washed twice in cold PBS, pelleted, snap-frozen, and stored at −80 °C. Pellets were thawed and 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) with ∼1 ml ceramic beads on a Fastprep-24 (MP Biomedicals). The entire lysate was then collected and adjusted to 1 ml before sonication with a 1/8” microtip on a Q500 sonicator (Qsonica) for 8-16 minutes (cycles of 10 seconds on and 20 seconds off). The sample tube was held suspended in a −20 °C 80% ethanol bath to prevent sample heating during sonication. Cell debris was then pelleted, and the supernatant was retained for ChIP or input. For each ChIP reaction, 20 µl Protein G Dynabeads (Invitrogen) were blocked (PBS + 0.5% BSA, incubate 40 minutes at room temperature), pre-bound with 5 µl of antibody in PBS (incubate 40 minutes at room temperature), and washed 2x with PBS before being incubated with supernatant (4°C overnight). Dynabeads were then washed (5 minutes per wash) 2x in FA lysis buffer and 3x in high-salt FA lysis buffer (50 mM Hepes-KOH pH 8.0, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM PMSF) and DNA was then eluted in ChIP elution buffer (50 mM TrisHCl pH 7.5, 10 mM EDTA, 1% SDS) at 65 °C for 20 minutes. 15 µl of input was mixed directly with 115 µl of ChIP elution buffer for input samples. Crosslinks were then reversed (65 °C, 5hr) before treatment with RNAse A (37 °C, 1 hour) and then Proteinase K (65 °C, 2 hours). DNA was then purified using the ChIP DNA Clean & Concentrator kit (Zymo Research).
Indexed sequencing libraries were generated using the NEBNext Ultra II DNA Library Prep kit (NEB Cat # E7645), pooled, and then sequenced by paired-end (2x150bp) Illumina sequencing.
spike-in normalised ChIP-seq
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
Eluted Rpb1 ChIP DNA from a mixture S. cerevisiae (MS67) and C. glabrata (MS99). MS67 was arrested in G1 and collected at timepoint 4.
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| Data processing |
read alignment, ChIP-seq and gDNA data Bowtie (version 1.0.1)
read alignment, RNA-seq data STAR (version 2.5.3a
bigWig file generation custom code
Assembly: sacCer3
Supplementary files format and content: bigWig
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| Submission date |
Sep 11, 2023 |
| Last update date |
Nov 01, 2023 |
| 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 |
GPL33750 |
| Series (1) |
| GSE242874 |
RNA polymerase II dynamics and mRNA stability feedback scale mRNA amounts with cell size |
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| Relations |
| BioSample |
SAMN37351849 |
| SRA |
SRX21748511 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7773533_211117L28_exp211021_MS67_SCGE-T04_anti-Rpb1.bigWig |
26.4 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
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