![](/coreweb/template1/pix/main_left_bg.gif) |
![](/coreweb/template1/pix/pixel.gif) |
GEO help: Mouse over screen elements for information. |
|
Status |
Public on Nov 01, 2023 |
Title |
200808L04_exp200802_MS282_SCD-RAPA_input |
Sample type |
SRA |
|
|
Source name |
200808L04_exp200802_MS282_SCD-RAPA_input
|
Organisms |
Saccharomyces cerevisiae; Nakaseomyces glabratus |
Characteristics |
sample: Input ChIP DNA from a mixture S. cerevisiae (MS282) and C. glabrata (MS99). MS282 was treated with rapamycin before collection. cell type: yeast chip-antibody: n/a
|
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.
|
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
|
|
|
Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina NovaSeq 6000 |
|
|
Description |
Input ChIP DNA from a mixture S. cerevisiae (MS282) and C. glabrata (MS99). MS282 was treated with rapamycin before collection.
|
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
|
|
|
Submission date |
Sep 11, 2023 |
Last update date |
Nov 01, 2023 |
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 |
GPL33750 |
Series (1) |
GSE242874 |
RNA polymerase II dynamics and mRNA stability feedback scale mRNA amounts with cell size |
|
Relations |
BioSample |
SAMN37351487 |
SRA |
SRX21748406 |
Supplementary file |
Size |
Download |
File type/resource |
GSM7773895_200808L04_exp200802_MS282_SCD-RAPA_input.bigWig |
5.4 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector![Help](/coreweb/images/long_help4.gif) |
Raw data are available in SRA |
|
|
|
|
![](/coreweb/template1/pix/main_right_bg.gif) |