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Sample GSM3788396

Query DataSets for GSM3788396

Status

Public on Mar 03, 2020

Title

top2_4 H2A rep1

Sample type

SRA

Source name

Saccharomyces cerevisiae

Organism

Saccharomyces cerevisiae

Characteristics

strain: JB479
genotype: Mat a his4-539 ura3-52 top2-4
chip antibody: H2A (1:500, active motif, 39235)

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

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).

Extracted molecule

genomic DNA

Extraction protocol

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)). 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 H2AP (Abcam), and these were placed on a rotating wheel at 4°C for 15 – 20 h. For experiments where RPA1 ChIP was performed on the same sample, 75 ml starting cultures were used meaning supernatant could be split into 3X 2ml and 1 ml for input, with RPA1 antibody (1:10000, Agrisera) added to one 2 ml aliquot. For experiments where RPA1 ChIP was exclusively performed, 25 ml starting cultures were used to make a split of 1X 2ml for antibody addition and 1ml for input.
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 purification kit and eluted in 50 μl for H2A or H2AP or 40 μl for RPA1. DNA amount was measured using the Qubit 2.0 Fluorometer (Life technologies) as per the manufacturer’s instructions. For H2A or H2AP samples, libraries were prepared using the NEBnext Ultra II library kit (NEB) as per the manufacturer’s instructions. PCR enrichment required 13 cycles. PCR purification was carried out using AMPure XP beads. For RPA1 library preparation 34 ul from the RPA1 samples and 1 ng DNA in 34 ul water from the input were used. 5 µl 10 x NEB2.1 buffer and 5 µl of random primers (8N, 3 mg/ml stock) were added and the samples were boiled at 95°C for 5 minutes and immediately placed to ice for 5 minutes. 5 µl 10 x dNTP with dUTP instead of dTTP (2 mM each) and 1 µl T4 polymerase (NEB) were added and the mixture was incubated at 37°C in a thermal cycler for 20 min, and 5 µl 0.5 M EDTA (pH 8) was immediately added to stop the reaction. The resulting dsDNA was used to create libraries using the Ultra II library kit (NEB) as per the manufacturer’s instructions.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina MiSeq

Description

processed data file:
N180118_top2-4_to_H2A_SPMR_FE.bedgraph_binned_50_norm.wig

Data processing

FASTQ files were generated by Illumina basespace (https://basespace.illumina.com/home/index).
The resulting sequences were aligned to a reference genome (R64-1-1, Saccharomyces cerevisiae S288c assembly from Saccharomyces Genome Database) using Bowtie 2 generating a SAM output file for each sample (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml). Reads from MySeq were trimmed 25 bp from 3’ and 1 bp from the 5’ end, while reads from NextSeq were not trimmed. Command for MySeq reads: bowtie2 -p 14 -x [path to index folder] --trim3 25 --trim5 1 -1 [Path and name of R1 fastq file] -2 [Path and name of R2 fastq file] -S [name of the resulting .sam file] Command for NextSeq reads: bowtie2 -p 14 -x [path to index folder] --trim3 0 --trim5 0 -1 [Path and name of R1 fastq file] -2 [Path and name of R2 fastq file] -S [name of the resulting .sam file]
SAM files were then converted into sorted BAM files by using SAMtools (http://samtools.sourceforge.net/): samtools sort [name of the .sam file generated with bowtie2] -o [name for the resulting .bam file] -O bam -T [name for resulting .bam file wo .bam]
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. Example command: macs2 callpeak -t [sorted BAM file from yh2a data]-c [sorted BAM file from h2a data]-f BAMPE -g 12100000 -n [name for output file] -B -q 0.01 --SPMR
The data then was sorted into 50 bp bins and used for meta data analysis using custom made R programs.
Genome_build: R64-1-1, Saccharomyces cerevisiae S288c assembly from Saccharomyces Genome Database
Supplementary_files_format_and_content: wig

Submission date

May 21, 2019

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