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Sample GSM4320017

Query DataSets for GSM4320017

Status

Public on Mar 03, 2020

Title

degron background wt 80 min spiked input rep2

Sample type

SRA

Source name

Saccharomyces cerevisiae/Schizosaccharomyces pombe

Organisms

Schizosaccharomyces pombe; Saccharomyces cerevisiae

Characteristics

s. cerevisiae strain: JB12
s. cerevisiae genotype: Mat a ade2-1 his3-11 leu2-3 trp1-1 ura3-1 can1-100 UBR1::GAL1-10-Ubiquitin-M-LacI fragment-Myc-UBR1 (HIS3) leu2-3::pCM244 (CMVp-tetR’-SSN6, LEU2) x3 pRS316
s. pombe strain: AW507
s. pombe genotype: h-urg1::Purg1lox-HO, LEU-HOcs-his3+-λ-EU2, leu1-32, his3-D1
time: 80 min after alpha factor release
chip antibody: None

Treatment protocol

Cultures were fixed at 25°C in YP + 1% formaldehyde (Sigma) for 45 min. 125 mM glycine was then added for 5 min. Cells were washed with PBS before being pelleted and frozen in liquid nitrogen.

Growth protocol

Sacchamromyces cerevisiae top2-td cell cultures for alpha factor release experiments were prepared as described previously -S. A. Schalbetter, S. Mansoubi, A. L. Chambers, J. A. Downs, J. Baxter, Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability. Proceedings of the National Academy of Sciences. 112, E4565-70 (2015). Schizosaccharomyces pombe cells were induced for 2h as described in Adam T. Watson, Petra Werler, Antony M. Carr, Regulation of gene expression at the fission yeast Schizosaccharomyces pombe urg1 locus, Gene, Volume 484, Issues 1-2, 2011, Pages 75-85, ISSN 0378 1119, https://doi.org/10.1016/j.gene.2011.05.028.

Extracted molecule

genomic DNA

Extraction protocol

S. cereviseae pellets from 50 ml culture were resuspended in 500 μl SDS buffer (1% SDS, 10 mM EDTA, 5M Tris HCl, cOmplete Tablets, Mini EDTA-free EASYpack (Roche), PhosSTOP (Roche)). Aliquots of S. pombe cell pellets were resuspended in 250 µl SDS buffer, and 1/1000 volume of the original S. cerevisiae culture (corresponding to 1:10 S. pombe to S. cerevisiae ratio) was added to each S. cerevisiae samples. Cells were lysed in a FASTPREP machine, 5 rounds of 1 min at 6.5 power, with 200 μl of 0.5 mm silica beads. Lysate was spun out and IP buffer (0.1% SDS, 1.1% Triton-X-100, 1.2 mM EDTA, 16.7 mM TRIS HCl (pH8), cOmplete Tablets, Mini EDTA-free EASYpack (Roche), PhosSTOP (Roche)) was added to a final volume of 1 ml. Samples were sonicated using the Focused-Ultrasonicator (Covaris, M220) (Average incident power - 7.5 Watts, Peak Incident Power - 75 Watts, Duty Factor - 10 %, Cycles/Burst - 200, Duration - 20 min). The sample was centrifuged for 20 min at 13,000 rpm at 4°C. Supernatant was then diluted to 1:10 (5 ml total). 50 μl protein A Dynabeads (Invitrogen, 10002D) and 50 μl protein G Dynabeads (Invitrogen, 10004D), were washed 3 times in IP buffer followed by adding to the sample and incubating for 2 h at 4°C. Supernatant was split, with 2X 2 ml being taken to 15 ml Falcon tubes, and 1 ml being kept at -20°C as an input sample. To the two 2 ml samples antibody was added, either H2A 1:500 (active motif) or 1.6 μg/ml yH2A (Abcam), and these were placed on a rotating wheel at 4°C for 15 - 20 h. A preparation of Dynabeads (Invitrogen), Protein A (30 μl) and Protein G (30 μl), was washed 3 times in IP buffer. This was added to each sample and incubated at 4°C for 4 h. Supernatant was removed and beads were washed at 4°C for 6 min in TSE-150 (1% Triton-X-100, 0.1% SDS, 2 mM EDTA, 20 mM Tris HCl (pH8), 150 mM NaCl), followed by TSE-500 (1% Triton-X-100, 0.1% SDS, 2 mM EDTA, 20 mM Tris HCl (pH8), 500 mM NaCl), followed by LiCl wash (0.25 M LiCl, 1% NP-40, 1% dioxycholate, 1 mM EDTA, 10 mM Tris HCl (pH8)) and finally Tris-EDTA (TE pH8). Elution was carried out in 400 μl elution buffer, for 30 min at room temperature. At the same time 50 μl from the input sample was added to 150 μl of elution buffer. 20 μl of 5 M NaCl and 10 μl of 10 mg/ml proteinase K (Invitrogen) was then added to the input, and 40 μl and 20 μl to the IP samples respectively. These were incubated at 65°C overnight. Then 10 μl of DNase-free RNase (Roche) was added to the input and 20 μl to the IP samples, and they were left at 37°C for 30 min. All DNA was purified with a Qiagen PCR pufirication kit and eluted in 50 μl. DNA amount was measured using the Qubit 2.0 Fluorometer (Life technologies) as per the manufacturer’s instructions.
Libraries were prepared using the NEBnext Ultra II library kit (NEB) as per the manufacturers instructions. PCR enrichment required 13 cycles. PCR purification was carried out using AMPure XP beads.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina NextSeq 500

Description

wt_N191127_80_spike_norm.wig

Data processing

FASTQ files were generated by Illumina basespace (https://basespace.illumina.com/home/index).
The resulting sequences were aligned to S. cerevisiae reference genome (R64-1-1, Saccharomyces cerevisiae S288c assembly from Saccharomyces Genome Database) and to S. pombe (Downloaded from https://www.pombase.org/downloads/genome-datasets (9/4/2018)) using Bowtie 2 generating a SAM output file for each sample (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml).
Unaligned reads from S. cerevisiae were aligned to S. pombe genome and unaligned reads from S. cerevisiae were aligned to S. pombe genome to aquire unique reads using samtools (http://samtools.sourceforge.net/) and bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml)
SAM files were then converted into sorted BAM files by using SAMtools (http://samtools.sourceforge.net/):
BAM files were used for Model-based Analysis of ChIP-Seq (MACS2). We used the ‘call peak’ function which also generates genome wide score data. These were used to generate fold enrichment tracks.
The data then was sorted into 50 bp bins and used for meta data analysis using custom made R programs
H2AP enrichment data over H2AP was normalized by multiplying each value by OR= Enrichment over input in S. cerevisiae genome/enrichment over input in S. pombe genome
Genome_build: R64-1-1, Saccharomyces cerevisiae S288c assembly from Saccharomyces Genome Database
Genome_build: Schizosaccharomyces pombe (pombase (9/4/2018))
Supplementary_files_format_and_content: wig files contained normalised realtive H2AP enrichment over H2A

Submission date

Feb 19, 2020

Last update date

Mar 04, 2020

Contact name

Andrea Keszthelyi

E-mail(s)

ak483@sussex.ac.uk

Organization name

University of Sussex

Department

Genome Damage and Stability Centre