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| Status |
Public on Jul 21, 2021 |
| Title |
FigS3F_22009_75IP |
| Sample type |
SRA |
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| Source name |
Saccharomyces cerevisiae cells
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
sac_cer strain: sac_cer strain: K22009
can_gla strain: can_gla strain: K23308
antibody: anti-PK antibody (Bio-Rad)
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| Treatment protocol |
none
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| Growth protocol |
Yeast cells were grown in YPD to reach a density of 0.3-0.6 OD600.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
To crosslink cells, 45ml of yeast culture was mixed with 4.2ml of fixation solution (50mM Tris-HCl pH 8.0, 100mM NaCl, 0.5mM EGTA, 1mM EDTA, 30% Formaldehyde) and incubated at 18°C for 30 minutes. The crosslinking reaction was quenched by incubating with 2ml of 2.5M Glycine for 5 min. Fixed cells were harvested, washed with ice-cold PBS and re-suspended in 1ml of ChIP lysis buffer (50mM Hepes-KOH pH 8.0, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate, 1mM PMSF, Roche protease inhibitor). For each ChIP, 10 OD600 units of Saccharomyces cerevisiae cells mixed with 5 OD600 units of Candida glabrata cells were pelleted and re-suspended in 0.3ml of ChIP lysis buffer. Cells were mixed with glass beads and disrupted by FastPrep®-24 (MP Biomedicals, USA). The entire lysis was collected and sonicated at 4°C for 40 minutes using a Bioruptor (Diagenode, Belgium). The cell debris were removed by centrifugation and supernatants containing sheared chromatin with a size range from 100-800bp were adjusted to a final volume of 1ml with ChIP lysis buffer. Extracts were pre-cleared for 1 hour at 4°C with 30µl of Protein G Dynabeads (Invitrogen). 80µl of supernatant was taken as whole cell extract (W) and stored at -80°C. Five µg of anti-PK antibody (Bio-Rad) and 50 µl of Protein G Dynal beads (Invitrogen) was used for immunoprecipitation (6 hours-overnight, rotation at 4°C). The beads were subsequently washed for 5 minutes with the following buffers: 2×ChIP lysis buffer; 3x ChIP high-salt lysis buffer (50mM Hepes-KOH pH 8.0, 500mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Sodium deoxycholate, 1mM PMSF); 2×ChIP wash buffer (10mM Tris-HCl pH 8.0, 0.25M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1mM EDTA, 1mM PMSF) and 1x TE buffer (10mM Tris-HCl pH 8.0, 1mM EDTA, 50mM NaCl). The immunoprecipitated chromatin was eluted by incubation of beads with 120µl of TES buffer (50mM Tris-HCl pH 8.0; 10mM EDTA; 1% SDS) at 65°C for 15 minutes. The supernatants were collected and termed the IP sample. The whole cell extract sample (W) was mixed with 40µl of TES3 buffer (50mM Tris-HCl pH 8.0; 10mM EDTA; 3% SDS). Both samples were de-crosslinked at 65°C overnight. RNA was degraded by incubating with 2-µl of RNAse A (10mg/ml, Roche) at 37°C for 1 hour and protein was subsequently removed by incubation with 10µl of Proteinase K (18mg/ml, Roche) at 65°C for 2 hours. DNA was purified using ChIP DNA Clean & Concentrator kit (Zymo Research, USA).
NEBNext Fast DNA Library Prep set for Ion Torrent, E6270
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Ion Torrent Proton |
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| Description |
Arrested in G2 (Nocodazole) at 37˚C
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| Data processing |
Library sequencing was carried out on the Ion Torrent Proton
The quality of the raw sequence data was examined using FastQC and trimming of reads was carried out using fastx_trimmer
Any reads smaller than 50bp were removed using ‘Filter FASTQ’
The final reads were aligned to the Saccharomyces cerevisiae genome (sacCer3, SGD) or Candida glabrata genome (CBS138, genolevures) using Bowtie2 with the default (--sensitive) parameters
To generate an alignment in which all the sequences exclusively align to the Saccharomyces cerevisiae genome, the whole reads were first aligned to Candida glabrata genome and the unaligned reads were retrieved as a separate Fastq file. Subsequently, these unaligned reads were re-aligned to Saccharomyces cerevisiae genome and the resulting aligned BAM file therefore contained reads that were unique to Saccharomyces cerevisiae. A same strategy was used to generate alignments unique to Candida glabrata.
Genome_build: SacCer3 or Candida glabrata (ASM254v2)
Supplementary_files_format_and_content: bigwig files
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| Submission date |
Feb 23, 2021 |
| Last update date |
Jul 21, 2021 |
| Contact name |
Kim Nasmyth |
| E-mail(s) |
kim.nasmyth@bioch.ox.ac.uk
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| Organization name |
University of Oxford
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| Department |
Biochemistry
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| Lab |
Nasmyth Lab
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| Street address |
South Parks Road
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| City |
Oxford |
| ZIP/Postal code |
OX1 3QU |
| Country |
United Kingdom |
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| Platform ID |
GPL18249 |
| Series (1) |
| GSE167318 |
Folding of cohesin's coiled coil is important for Scc2/4-induced association with chromosomes |
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| Relations |
| BioSample |
SAMN18037166 |
| SRA |
SRX10155700 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5101854_FigS3F_22009_75IP.bigwig |
27.5 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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