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Sample GSM3509916

Query DataSets for GSM3509916

Status

Public on Apr 17, 2019

Title

TOP2_ACF_ULS1_R2_IN

Sample type

SRA

Source name

Budding yeast

Organism

Saccharomyces cerevisiae

Characteristics

affinity tag: HA
treatment: ACF
chip antibody: none

Treatment protocol

exposed to 0, 100uM (Uls1 ChIP) or 250uM (Top2 ChIP) acriflavine (ACF) for 2 hours, before crosslinking with 1% formaldehyde for 10 mins and quenched in 125mM glycine for 10 mins

Growth protocol

yeast grown in 50ml YPD rich media to exponential phase

Extracted molecule

genomic DNA

Extraction protocol

To each yeast pellet add 300μl homogenization beads (0.5mm diameter, Thistle Scientific 11079105) and 200μl lysis buffer (50mM Hepes pH 7.5, 140mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate, protease inhibitors). Bead beat 5 x 30 seconds at power setting 6.5 (FastPrep), placing samples on ice for 5 minutes between each cycle. Pierce tube with a 26G needle and place tube inside a 5ml u-shaped tube, spinning lysis material into U-shaped tube by centrifugation at 2,000 rpm for 2 minutes at 4°C. Re-suspend any pelleted material and dilute in a further 300μl lysis buffer, taking this to a new Eppendorf tube. Centrifuge at 15,000 rpm for 15 minutes at 4°C, remove supernatant and resuspend pellet in 300μl lysis buffer, transferring to a 1.5ml Bioruptor tube (Diagenode, C30010016). Shear chromatin using Bioruptor, 10 cycles of 30s on/off (DNA should be sheared to fragments of 250-500bp). Centrifuge at 8,000 rpm for 5 minutes at 4°C. 60μl of the supernatant is then taken as input (40μl for DNA purification, 20μl to check protein levels), with the rest being added to antibody bound beads which are incubated for 3 hours at 4°C. Beads are washed in 1ml lysis buffer twice for 5 minutes and 1ml wash buffer once for 5 minutes (100mM Tris pH 8, 250mM NaCl, 0.5% NP-40, 0.5% Na-deoxycholate, 1mM EDTA, protease inhibitors) before elution in 60μl TE/1% SDS at 65°C for 15 minutes. Magnetic beads are collected using a magnetic rack, and supernatant removed to a new tube. 20μl is used to check protein levels, and 40μl kept for DNA purification. 1% SDS is added to input, 0.5μl RNase A (10mg/ml) is added to both input and IP DNA, and both samples are un- crosslinked overnight at 65°C in a PCR machine. 0.5μl Proteinase K (20mg/ml) is added after uncrosslinking and samples incubated for 1 hour at 65°C. DNA is purified using Quaigen QIAquick PCR purification kit (Quaigen, 28106) as per specifications, eluting in 50μl H2O.
Due to the low quantity of DNA present after immunoprecipitation, to ensure there is enough sample DNA for amplification and sequencing two experimental replicates are combined before ChIP-seq library preparation. Step 1 of library preparation allows repair of DNA ends. To 80μl pooled DNA, add 20μl MMX1 (1x T4-ligase buffer with ATP, 0.4mM dNTPs, 15 units T4 DNA polymerase, 10 units Klenow DNA polymerase, 30 units T4 polynucleotide kinase) and incubate for 60 minutes at 20°C. DNA is purified by addition of a 1:1 v/v of AMPure XP magnetic beads, incubating for 5 minutes before washing twice with 200μl 70% EtOH using a magnetic rack. Residual EtOH is removed before elution of DNA in 41μl H2O. Step 2 of library preparation adds an additional adenine nucleotide to DNA ends to which adapters will later be ligated. To 41μl DNA, add 9μl MMX2 (1x Klenow buffer, 2mM dATP, 15 units Klenow exo-), incubating for 30 minutes at 37°C. DNA is purified as in step 1 using a 1:1 v/v of Agencourt AMPure XP magnetic beads, eluting in a final volume of 20μl H2O. Step 3 of library preparation ligates adapters onto DNA which are used later as a template for PCR amplification of ChIPed DNA fragments to generate the final tagged DNA library. Adapter is ordered as two oligonucleotides (HFO424/425, Illumina paired end adapter sequence). HFO425 is phosphorylated as per manual specifications using T4 polynucleotide kinase (Thermo Fisher EK0032) before annealing to HFO424 by mixing an equimolar ratio of the two oligos and heating to 95°C for 5 minutes, reducing the temperature by 5°C every 5 minutes until reaching 25°C. Adapter is then run into a 1% agarose gel, gel purified and stored at -20°C before use. To ligate adapter to DNA, to 20μl DNA add 30μl MMX3 (8nM adapter, 1x T4 DNA ligase buffer with ATP, 400 units T4 DNA ligase), incubating overnight at room temperature. DNA is purified as in step 1 with a 1:1 v/v of Agencourt AMPure XP magnetic beads, eluting in 20μl H2O. Step 4 of library preparation amplifies the DNA samples using primers complimentary to the adapters now ligated onto each DNA molecule, with each primer also containing an additional unique sequence tag which is used to identify each sample after sequencing of the DNA. Details of the primers used can be found in Table 6–6. For each sample, 3 PCR reactions are set up containing 3μl DNA and 47μl MMX4 (1X HF buffer, 3 units Phusion DNA polymerase, 0.3μM oligos (HFO426 and sequencing primer), 0.4μM dNTPs, 200mM Trehalose). The PCR is carried out by denaturing for 3 mins at 98°C before 16-20 cycles of amplification (98°C 15 secs, 60°C 25 secs, 68°C 1 min) and a final step at 68°C for 5 mins. The three PCRs are then pooled and ran on a TAE gel containing 1% agarose and 0.5μg/ml EtBr. Once the gel has ran long enough to separate free adapter from amplified DNA, bands are cut from the gel at a size of 600bp and under, avoiding contamination with adapter. DNA is purified from the gel using Quaigen MiniElute columns (Quaigen, 28006) as per kit specifications except gel was melted at 37°C, eluting from the column in 10μl EB. DNA concentration was quantified via Qubit using 1:199 DNA:Qubit dsDNA High Sensitivity assay working solution (Invitrogen, Q32854, as per kit specifications). DNA quality and mean fragment size were checked on a TapeStation, running samples onto D1000 ScreenTape with D1000 reagents (Agilent, 5067-5582, 5067-5583).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina Genome Analyzer

Data processing

Reads were mapped to the S. cerevisiae W303 genome (Matheson et al., 2017) using BWA (Li and Durbin, 2010) with default parameters for paired end sequences
Indexed BAM files were quality filtered using samtools view (Li et al., 2009) with a quality filter of 28 and a -F FLAG value of 1796. QualiMap (Okonechnikov et al., 2016) was used to check the quality of alignment in both the unfiltered and quality filtered BAM files. Replicate correlation was checked using Pearson’s correlation, calculated using deepTools multiBamSummary and plotCorrelation (Ramírez et al., 2016). Replicates or inputs (within one genotype) were pooled using samtools merge with default parameters. The resulting file was then sorted using samtools sort and indexed using samtools index, all with default parameters (Li et al., 2009).
Peak calling was carried out using MACS2 subcommands (Zhang et al., 2008). PCR duplicates were filtered using macs2 filterdup. IP pileup track was generated using macs2 pileup, extending reads to the average fragment size. IP pileup track was generated using macs2 pileup, extending reads to the average fragment size. INP local lambda track was generated using macs2 pileup (-B), generating three tracks where reads were extended in both directions by half the average fragment size (termed “d”), 500bp (termed “1kb_slocal”), or 2500bp (termed “5kb_llocal”). 1kb_slocal and 5kb_llocal were normalised to “d” sized fragments using macs2 bdgopt (-m multiply). All tracks were combined to generate “local lambda” background track using macs2 bdgcmp (-m max), also normalising to maximum background noise (number of reads in INP x average fragment length / genome size) using macs2 bdgopt (-m max). IP and local lambda were normalised to counts per million (CPM) using macs2 bdgopt (-m multiply). A p-value statistical track was generated using macs2 bdgcmp (-m ppois). Peak calling was carried out using macs2 bdgpeakcall with a p-value cut-off of 0.1. IP/INP fold enrichment tracks were generated using macs2 bdgcmp.
Differential analysis compares signal at peak regions between different datasets. To do this, the peaks called in all Top2/Uls1 datasets must be combined to give a list of the regions at which we will be completing differential analysis. This is completed using bedtools intersectBed and mergeBed (Quinlan and Hall, 2010).
Transcription start site analysis. Analysis was carried out using the R based packages rtracklayer, ChIPseeker and Genomic Features (Lawrence, 2013; Lawrence et al., 2009; R Core Team, 2018; Yu et al., 2015). The input files for this analysis were our MACS2 peak files.
Repetitive region analysis. This analysis uses unfiltered reads where reads which map to multiple regions have not been removed. This analysis is slightly difficult in that you cannot use signal height to define whether the ChIPed protein is binding or not as the more repetitive the sequence, the higher the signal will be. However, if a pairwise comparison of two datasets within a repetitive region is carried out and one dataset shows significantly higher enrichment than the other, then it can be logically assumed that there is enrichment of the ChIPed protein within the “higher” dataset. Analysis was carried out at subtelomeric loci (+/- 5kb from each chromosome end), tRNA (as within genome reference), TKP/TY transposons (as within genome reference), Y’ elements (as within genome reference) and the rDNA locus. We identified a partial fragment of the rDNA locus within our W303 reference by using blastn (Altschul et al., 1990) to search the sequence for a single rDNA repeat from within chromosome 12 in the S288C reference (Engel et al., 2013), aligning to the sequence of chromosome 12 within our W303 reference. According to SGD (Stanford University, 2012), the sequence of a single rDNA repeat within the S288C reference is on Chromosome 12 at 459,797-468,931. The top hit from this blast search within our W303 reference is on Chromosome 12 at 478,018-478,813, containing the sequence of part of the 35S rDNA locus. First, a .bed file was generated containing the coordinates of all the regions within one repetitive region, for instance the 141 tRNA genes within our W303 reference. This was made within excel and saved as a tab delimited file. Data tracks were then generated with signal extracted at each repetitive region using intersectBed (Quinlan and Hall, 2010) and a peak list containing the SUM score under each peak in each dataset using the following commands for each dataset:
Genome_build: A whole-genome sequence for the W303 strain of S. cerevisiae was published by Matheson et al. (2017), containing 16 nuclear chromosomes, the mitochondrial genome and additional plasmids present in the sequenced strain. We were unable to obtain a copy of the Matheson genome annotation so generated our own. Initial annotation was carried out by inputting the W303_LYZE genome sequence (Genbank GCA_002163515.1, Assembly name ASM216351v1) into the Yeast Genome Annotation Pipeline (YGAP, Proux-Wéra et al., 2012). We used this as a basis for our genome annotation, using BLASTn (Altschul et al., 1990) to identify any ORFs which were not annotated by YGAP. YGAP identified 5634 ORFs, with 6600 present in the S288C reference, the gold standard of genome references in yeast (Saccharomyces Genome Database, version R64-2-1, Engel et al., 2013). 141 tRNA genes were identified, with 299 being present in the S288C reference. Where an unidentified gene had high similarity to Y prime helicase elements, this was labelled as “Y’ element” (54 items), and where it had high similarity to gag pol genes, this was labelled as “TKP/TY” (86 items). Any elements that could not be identified were labelled as “unknown” (4/5634 ORFs). Gene lengths were checked against S288C and a note was made for each gene as to whether they were the same or not, with 524/5634 differing in size in W303 compared to S288C. Additional information for each ORF was extracted from the YGP S288C reference using excel, including gene names, aliases and gene ontology information. ARS were identified using Biopython BioSeqIO (Cock et al., 2009), searching for variants of the consensus sequence for ARS (ATTTATATTTA, TTTTATATTTA, ATTTATGTTTA, TTTTATGTTTA, ATTTATATTTT, TTTTATATTTT, ATTTATGTTTT, TTTTATGTTTT), finding 333 ARS. Centromeric sequences were identified by blasting the nucleotide sequence of the S288C CEN1-16. CEN5 was identified by blasting for conserved centromeric elements CDEI and CDEIII as it had little homology to S288C CEN5.
Supplementary_files_format_and_content: bedgraph files. IP pileup track was generated using macs2 pileup, extending reads to the average fragment size. INP local lambda track was generated using macs2 pileup (-B), generating three tracks where reads were extended in both directions by half the average fragment size (termed “d”), 500bp (termed “1kb_slocal”), or 2500bp (termed “5kb_llocal”). 1kb_slocal and 5kb_llocal were normalized to “d” sized fragments using macs2 bdgopt (-m multiply). All tracks were combined to generate “local lambda” background track using macs2 bdgcmp (-m max), also normalizing to maximum background noise (number of reads in INP x average fragment length / genome size) using macs2 bdgopt (-m max). IP and local lambda were normalized to counts per million (CPM) using macs2 bdgopt (-m multiply).

Submission date

Dec 12, 2018

Last update date

Apr 17, 2019

Contact name

Helder Ferreira

E-mail(s)

hcf2@st-andrews.ac.uk

Organization name

University of St Andrews

Department

Biology