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Status |
Public on Mar 03, 2020 |
Title |
top2-td 120 min spiked H2A rep1 |
Sample type |
SRA |
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Source name |
Saccharomyces cerevisiae/Schizosaccharomyces pombe
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Organisms |
Schizosaccharomyces pombe; Saccharomyces cerevisiae |
Characteristics |
s. cerevisiae strain: JB13 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 top2-td TOP2 5’ upstream -100 to -1 replaced with kanMX-tTA (tetR-VP16)-tetO2 - Ub - DHFRL80P-ts - Myc -linker) pRS316 s. pombe strain: AW507 s. pombe genotype: h-urg1::Purg1lox-HO, LEU-HOcs-his3+-λ-EU2, leu1-32, his3-D1 time: 120 min after alpha factor release chip antibody: H2A (1:500, active motif, 39235)
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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.
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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.
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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.
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Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina NextSeq 500 |
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Description |
top2td_N191024_120_spike_norm.wig
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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
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Submission date |
Feb 19, 2020 |
Last update date |
Mar 04, 2020 |
Contact name |
Andrea Keszthelyi |
E-mail(s) |
ak483@sussex.ac.uk
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Organization name |
University of Sussex
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Department |
Genome Damage and Stability Centre
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Lab |
G4.15
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Street address |
University of Sussex, Sussex House, Falmer
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City |
Brighton |
ZIP/Postal code |
BN1 9RH |
Country |
United Kingdom |
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Platform ID |
GPL28173 |
Series (1) |
GSE131558 |
Cohesin causes replicative DNA damage by trapping DNA topological stress |
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Relations |
BioSample |
SAMN14134208 |
SRA |
SRX7748380 |
Supplementary data files not provided |
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Raw data are available in SRA |
Processed data are available on Series record |
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