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Sample GSM7099702

Query DataSets for GSM7099702

Status

Public on Mar 16, 2023

Title

6934_input (biol rep 2 control)

Sample type

SRA

Source name

yeast cell

Organism

Saccharomyces cerevisiae

Characteristics

strain: 6934
cell type: yeast cell
chip antibody: none
genotype: MATa ura3-52 trp1-289 leu2-3,112 bar1::LEU2 rad55:TRP1-PGAL1-RAD55

Treatment protocol

Cells at exponential phase were pre-grown in YP + raffinose to OD600=0.3 and synchronized with α-factor for 2.5 hours. Galactose was then added to the final concentration of 2% and cells were incubated at 30°C for another hour. After that, α-factor was washed away, cells were transferred to fresh YP + galactose media with nocodazole and incubated for 2 hours.
cells were crosslinked with 1.4% of formaldehyde at 30°C in a shaking water bath for 10 minutes. Crosslinking was stopped by adding glycine to the final concentration of 0.25 M and incubating the cultures for 5 minutes. 200 OD600 units were harvested for ChIP-seq for each sample.

Extracted molecule

genomic DNA

Extraction protocol

The 200 OD600 units harvested for each strain were split into four Eppendorf tubes (50 OD600 units in each) and resuspended in 400 µl of bead-beating buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 cOmplete Mini, EDTA-free Protease Inhibitor Cocktail tablet (Roche, 11836170001) per 5 ml of buffer). An equal volume of glass beads (0.5 mm diameter, BioSpec Products) was added, and the tubes were vortexed for 25 cycles (3,000 rpm; 30 seconds of vortexing, 1 minute of resting on ice). After vortexing, cells were separated from the beads and cells and nuclei were lysed with 0.5% (w/v) SDS for 10 minutes on ice. Tubes were then centrifuged at 20,817 g for 15 minutes at 4°C, the resulting supernatants were removed, the pellets were washed with 1 ml of 0.1% SDS buffer (50 mM HEPES-KOH pH 7.5, 150 mM NaCl, 0.1% (w/v) SDS, 1% Triton X-100 (w/v), 0.1% Sodium Deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 cOmplete Mini, EDTA-free Protease Inhibitor Cocktail tablet (Roche, 11836170001) per 5 ml of buffer) and resuspended in 300 µl of the same buffer. Cell extracts were then sonicated in Bioruptor Plus sonicator (Diagenode) for 20 cycles (30 seconds ON / 30 seconds OFF) on the HIGH power setting. After the sonication, cell debris was spun down at 20,817 g for 15 minutes at 4°C, and supernatants were transferred into fresh Eppendorf tubes containing 1 ml of the 0.1% SDS buffer. Samples belonging to the same strain (split into 4 Eppendorf tubes at the beginning) were then pooled together. 10 µl were collected as input aliquots for each sample, mixed with 390 µl of TE (10 mM Tris-HCl, pH 8, 1 mM EDTA) and stored at -80°C. Four immunoprecipitation reactions were set up for each sample by mixing 1 ml of sonicated cell lysate with 15 µl of Anti-Rad51 (y-180) rabbit polyclonal (Santa Cruz Biotechnology sc-33626) antibody and 15 µl of Dynabeads protein A magnetic beads (Invitrogen, 10002D). The mixtures were incubated overnight at 4°C on a rotating wheel. Next day, beads were recovered using a magnet and sequentially washed with wash buffer 1 (50 mM HEPES-KOH pH 7.5, 275 mM NaCl, 0.1% (w/v) SDS, 1% Triton X-100 (w/v), 0.1% Sodium Deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 cOmplete Mini, EDTA-free Protease Inhibitor Cocktail tablet (Roche, 11836170001) per 5 ml of buffer), wash buffer 2 (50 mM HEPES-KOH pH 7.5, 500 mM NaCl, 0.1% (w/v) SDS, 1% Triton X-100 (w/v), 0.1% Sodium Deoxycholate, 1 mM EDTA, 1 mM PMSF, 1 cOmplete Mini, EDTA-free Protease Inhibitor Cocktail tablet (Roche, 11836170001) per 5 ml of buffer), wash buffer 3 (10 mM Tris-HCl, pH 8, 0.25 M LiCl, 1mM EDTA, 0.5% IGEPAL, 0.5% Sodium Deoxycholate) and wash buffer 4 (10 mM Tris-HCl, pH 8, 1 mM EDTA) for 5 minutes each wash on a rotating wheel at room temperature. To elute precipitated chromatin, beads belonging to the same sample were pooled together in 200 µl of TES (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 1% SDS) and then incubated at 65°C for 10 minutes with mixing. Tubes were then vortexed, incubated at 65°C for additional 10 minutes and the eluted chromatin was separated from the magnetic beads by centrifugation at 15,871 g for 3 minutes. The beads were washed with 200 µl of TE on the rotating wheel for 15 minutes at room temperature. The tubes were then centrifuged at 15,871 g for 3 minutes, and the resulting supernatants were mixed with the corresponding aliquots of the eluted chromatin. To remove proteins from the IPed DNA, Proteinase K was added to the immunoprecipitated chromatin samples to the final concentration of 1 mg/ml, and the tubes were incubated at 65°C overnight. Next day, immunoprecipitated DNA was purified using QIAquick® PCR Purification Kit (Qiagen, 28106) and eluted in 35 µl of water.
ChIP-seq libraries were prepared using DNA SMARTTM ChIP-Seq Kit (Takara Bio, 634865) according to the manufacturer’s instructions
12 samples were indexed as indicated in the sample description. Indexed samples were pooled and sequences together.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina MiniSeq

Description

indexes used for this sample - R9 (CATAGCCG) and F2 (ATAGAGGC)
mutant_7208_7208_control_6933_6934_ratio_mean.bigwig

Data processing

Reads in files S0_L001_R1_001 and S0_L001_R2_001 were demultiplexed using Barcode Splitter (Galaxy Version 1.0.1). The barcode sequences used for this are indicated in the sample description. Number of allowed mismatches: 0; Number of allowed barcodes nucleotide deletions: 0.
Reads were filtered for quality and poly-A tails added during library preparation using DNA SMARTTM ChIP-Seq Kit (Takara Bio, 634865) were removed using RINSEQ (Galaxy Version 0.20.4+galaxy1). Minimum mean score threshold to conserve sequences: 15; Maximal N percentage threshold to conserve sequences 2; Minimum length of poly-A/T to trim at the 5'-end: 8; Trim sequence by quality score from the 3'-end -> Quality score threshold to trim positions: 20 -> Type of quality score calculation to use: Minimum -> Rule to use to compare quality score to calculated value: Less than -> Size of the sliding window used to calculated quality score by type: 1 -> Step size used to move the sliding window: 1.
Adapter sequences were removed using Trim Galore! (Galaxy Version 0.6.7+galaxy0). Adapter automatic detection and default settings were used.
Paired reads were interleaved using seqtk_mergepe (Galaxy Version 1.3.1) using default parameters.
Processed and interleaved reads were aligned to sacCer3 reference genome using Bowtie2 (Galaxy Version 2.4.2+galaxy0) and default settings.
Reads from aligned IP samples were normalised to those from aligned input samples using bamCompare (Galaxy Version 3.3.2.0.0). Method to use for scaling the largest sample to the smallest: read count; How to compare the two files -> Compute the ratio of the number of reads -> Pseudocount: 10 10.
Rad51-ChIP signal in PGAL1-RAD55 srs2Δ rad54Δ mutants was compared to PGAL1-RAD55 controls and in induced (GAL) NK1325 cells to uninduced (RAF) NK1325 cells using bigwigCompare (Galaxy Version 3.3.2.0.0) by computing the ratio signals using default settings. The average signal ratio for the two biological repeats was computed using bigwigCompare (Galaxy Version 3.3.2.0.0) -> compute the mean of the signal in the two files.
Assembly: reference sequence sacCer3
Supplementary files format and content: format: bigwig; content: Rad51 ChIP-seq signal comparison between mutant (treated) and control

Submission date

Mar 15, 2023

Last update date

Mar 17, 2023

Contact name

Tadas Andriuskevicius

Organization name

The University of Edinburgh

Department

Institute of Cell Biology, School of Biological Sciences

Street address

Alexander Crum Brown Road