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| Status |
Public on Dec 01, 2024 |
| Title |
WG215_YPD_rep2 |
| Sample type |
SRA |
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| Source name |
W303-1a
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strain: W303-1a
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| Growth protocol |
For all experiments, cells were grown overnight to saturation in 4 mL YPD medium at 30 °C with shaking at 250 rpm, then diluted to OD600 0.5 in 500 mL YPD, or YP-Raffinose (for galactose induction experiments), and grown until OD600 2.0 in a 2 L baffled flask with antifoam 204 (Sigma A8311) added to 0.0001%. Alpha mating factor was added to a concentration of 20 µg/mL, and the cultures were incubated for a further 90 min. A second dose of alpha factor was added to a final total concentration of 26.6 µg/mL (including first dose), and the cultures were incubated for a further 30 min. At this point >99% of the cells were arrested in G1.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
CC-seq-v2 is an update of our prior published method that improves sensitivity by several orders of magnitude, enabling the detection of physiological Top2ccs in etoposide-unchallenged cells. Transient physiological Top2ccs were rapidly fixed by denaturation via the direct addition of 70 mL of culture to 150 mL of ethanol (-20˚C) in a 250 mL centrifuge vessel (NALGENE), which had been pre-chilled overnight at -20˚C. Mixtures were immediately vigorously shaken, resulting in a final concentration of 70% ethanol at ~0˚C, prior to incubation at -20˚C for 15 min. Tubes were centrifuged at 3500g for 3 min (4˚C) and the supernatant was aspirated fully. Cell pellets were resuspended in 1.2 mL spheroplasting buffer (1 M sorbitol, 50 mM NaHPO4 buffer pH 7.2, 10 mM EDTA) containing 400 μg/mL Zymolyase 100 T (AMS Biotech) and 1% β-mercaptoethanol (Sigma) for 20 min at 37 °C. The entire spheroplast suspension was transferred to 5 mL eppendorfs containing 3.773 ml of ethanol (-20˚C), shaken vigorously to mix, and pelleted by centrifugation at 1000g for 2 min. The ethanolic supernatant of each sample was fully aspirated prior to addition of 800 µL 1× STE buffer (2% SDS, 0.5 M Tris pH 8.1, 10 mM EDTA, 0.05% bromophenol blue). Cells were lysed using a small plastic pestle (VWR) before addition of a further 1600 µL 1× STE buffer, with mixing by inversion and incubation for 10 min at 65 °C. Samples were cooled on ice and 1250 µL Phenol-Chloroform-isoamyl alcohol (25:24:1; Sigma) was added. The mixtures were emulsified by shaking vigorously 30 times, prior to incubation at room temperature (RT) for 5 min. The mixtures were re-emulsified by shaking vigorously 30 times, prior to phase separation by centrifugation at 20,000 × g for 5 min. 2 mL of the aqueous phase was removed to a clean microcentrifuge tube, taking care not to disturb the interphase, and nucleic acids were precipitated with 3.5 mL ice-cold ethanol, pelleted by centrifugation, washed with 5 mL ice-cold 70% ethanol, and dissolved in 1 mL 1x TE buffer pH 7.5 overnight at 4 °C. 1 mL aliquots of nonproteolysed DNA (prepared as above) were fully resuspended by heavy vortexing and inversion on a rotation wheel for 15 min (RT), prior to sonication to an average fragment size of 300–400 bp with Covaris (duty cycle: 10%, intensity/peak power incidence: 75 W, cycles/burst: 200, time: 21 min). A sample of 12 uL was removed and incubated at 37˚C for 45 min with 3 uL of RNAse A (50 mg/mL). DNA shearing was checked by 1% agarose gel electrophoresis, and DNA was quantified in technical triplicate with the Qubit (ThermoFisher) and High Sensitivity reagents. Human-Lambda DNA spike was added to 950 uL of sample, then the total volume was made up to 1 mL with 1x TE (pH 7.5). Triton-X100, N-Lauroylsarcosine sodium salt and NaCl were then added to complete the binding buffer (final concentrations: 0.3 M NaCl, 0.2% Triton-X100, 0.1% N-Lauroylsarcosine sodium salt). Each sample was divided over two QIAquick silica fibre membrane spin columns (QIAGEN). Under these conditions, protein, but not nucleic acids, bind to the silica membrane, leading to selective retention of any CCs. The flowthrough was reapplied to the column to improve yield. Columns were washed six times with 600 µL of TEN (10 mM Tris pH 7.5, 1 mM EDTA, 0.3 M NaCl) per 1 min wash to remove any residual non-CC DNA fragments, prior to elution twice with 125 µL TES (10 mM Tris pH 7.5, 1 mM EDTA, 0.5% SDS). The SDS detergent in the elution buffer releases interactions between protein and silica, thereby releasing bound CCs.
Eluted products were pooled to 500 µL in TES and incubated with 1 mg/mL Proteinase K (Sigma) for 60 min at 50 °C, prior to overnight ethanol precipitation at −20 °C with 1.41 mL ethanol, 0.2 mg/mL glycogen and 200 mM NaOAc. The DNA-glycogen precipitate was pelleted by centrifugation at 20,000 × g for 1 h at 4 °C, washed once with 1.5 mL 70% ethanol without disturbing the pellet, and re-centrifuged for 15 min. The supernatant was aspirated, and the pellet was air-dried for 10 min at room temperature, prior to solubilisation in 26 µL 10 mM Tris-HCl pH 7.5. DNA concentration was measured in a 1 µL sample using a Qubit (ThermoFisher) and High Sensitivity reagents. The remaining 25 µL was used as input for one round of end repair and adapter ligation with NEBNext Ultra II DNA Library Preparation kit (NEB), according to manufacturer’s instructions, except all steps were conducted at half-scale and a custom P7 adapter was used. The use of custom adapters is to allow differentiation of the sheared end (P7 adapter) from the Top2cc end (P5 adapter). After ligation of the P7 adapter, TE was added to a final volume of 90 uL. DNA was isolated with AMPure XP beads (Beckman Coulter) according to manufacturer’s instructions (beads:input of 78:90 uL) and eluted in 85 µL water. Next, 10 uL of 10X Cutsmart buffer and 5 uL Shrimp Alkaline Phosphatase (rSAP; NEB) were added before incubation for 30 min at 37˚C. DNA was isolated again with AMPure XP beads (Beckman Coulter) according to manufacturer’s instructions (beads:input of 83:100 uL) and eluted in 50 µL 10 mM Tris-HCl. Samples were diluted twofold with 50 µL TDP2 reaction buffer (100 mM TrisOAc, 100 mM NaOAc, 2 mM MgOAc, 2 mM DTT, 200 µg/mL BSA) and incubated with 3 µL of 10 µM recombinant zebrafish TDP2 catalytic domain (Addgene plasmid #200512) for 1 h at 37 °C. DNA was isolated again with AMPure XP beads (beads:input of 83:103) and eluted in 26 µL 10 mM Tris-HCl. Next, a second round of adapter ligation was conducted using a new custom adapter dp-P5 without prior end repair in order to prevent dephosphorylation of DNA 3′-phosphate termini which may be generated by TDP2 activity on 3′-CCs. After ligation of the dp-P5 adapter to the Top2-cleaved end, sample volume was adjusted to 90 uL with Tris-HCl, DNA was isolated with AMPure XP beads (beads:input of 78:90) and eluted in 16 µL 10 mM Tris-HCl. DNA concentration was measured in 1 µL using a Qubit. The remaining 15 µL was used as template for the NEBNext Ultra II PCR step using universal primer (P5 end) and indexed primers for multiplexing (P7 end), according to manufacturer’s instructions (12–15 cycles). PCR reactions were diluted with 50 µL 10 mM Tris-HCl, DNA was isolated with AMPure XP beads (beads:input of 83:100) and eluted in 30 µL 1 mM Tris-HCl pH 8.1, 100 uM EDTA. Sample DNA molarities were quantified using the Bioanalyzer or Tapestation (Agilent).
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Multiplexed library pools were sequenced on the Illumina NextSeq 500 (Kit v2–75 cycles) or Illumina NextSeq 1000 (Kit P2–100 cycles), with paired-end read lengths of 42, or 51 bp, respectively. Paired-end reads that passed filter were aligned using bowtie2 (options: -X 1000 –no-discordant –very-sensitive –mp 5,1 –np 0), using MAPQ0 settings, then SAM files processed via terminalMapper (Perl, v5.22.1; https://github.com/Neale-Lab) that computes the coordinates of the protein-linked 5′-terminal nucleotide. The reference genome used in this study is our in-house Cer3H4L2 (S. cerevisiae) build, which we generated by inclusion of the his4::LEU2 and leu2::hisG loci into the Cer3 yeast genome build. Specifically, Cer3H4L2 is identical to the sacCer3 reference genome (R64-1-1), with the addition of two ectopic insertions: 1,173 bp of hisG sequence inserted at the LEU2 locus at position 91,965, and 3,077 bp of LEU2 sequence including 77 bp of associated unidentified bacterial sequence at position 65,684. All subsequent analyses were performed in R (Version 4.4) using RStudio (Version 2024.04.2+764), unless indicated otherwise. Yeast data sets were filtered to exclude the rDNA, the CUP1-2 locus, the mitochondrial genome and the 2-micron plasmid. Final averaged datasets used for generating figures are included in the supplementary files for the series accession.
Assembly: Cer3H4L2
Supplementary files format and content: FullMap files containing sparsely-formatted, 1bp histograms detailing the total number of Watson (+) and Crick (-) hits for all mapped base-pairs. 4-column format (Chr, Pos, Watson, Crick).
Library strategy: Top2 CC-seq
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| Submission date |
Oct 23, 2024 |
| Last update date |
Dec 01, 2024 |
| Contact name |
William Gittens |
| E-mail(s) |
w.gittens@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 |
Neale Lab
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| Street address |
Science Park Road
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| City |
Brighton |
| State/province |
I am not in the U.S. or Canada |
| ZIP/Postal code |
BN19Rq |
| Country |
United Kingdom |
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| Platform ID |
GPL19756 |
| Series (1) |
| GSE280153 |
Osmotic disruption of chromatin induces Topoisomerase 2 activity at sites of transcriptional stress [CC-seq-v2] |
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| Relations |
| BioSample |
SAMN44406882 |
| SRA |
SRX26477220 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM8588938_FullMap.Cer3H4L2_WG215_YPD_rep2.txt.gz |
8.5 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
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