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| Status |
Public on Dec 01, 2020 |
| Title |
H2A.ZPht1_swr1∆ Rep3 |
| Sample type |
SRA |
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| Source name |
H2A.ZPht1_swr1∆
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
genotype: swr1{delta}
antibody: anti-myc (9B11 - Cell Signaling, Cat# 2276)
expressing: H2A.ZPht1-myc
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| Growth protocol |
S.pombe strains were grown in YES media. ~150x107 cells were harvested from o/n cultures and processed for ChIP
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde (Sigma) for 15 min at room temperature and lysed using a bead beater (Biospec Products). Cell lysates were sonicated in a Bioruptor (Diagenode) (30/40 min, 30 s On and 30 s Off at ‘High’ (200W) position). For CENP-ACnp1 ChIPs, anti-CENP-ACnp1 serum was used with Protein G sephrose beads (Roche). For all other ChIPs, Protein G Dynabeads (Life Technologies) were used along with anti-Myc 9B11 (Cell Signaling) or anti-Flag M2 antibody (Sigma), as appropriate. Immunoprecipitated DNA was recovered using Qiagen Min-Elute PCR purification kit.
ChIP-seq libraries were generated using an Illumina-based protocol with custom reagents using NEXTFlex-96 DNA Barcodes (Bioo Scientific). Briefly, immunoprecipitated DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt phosphorylated ends were treated with Klenow fragment (3’ to 5’ exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina adapters, which have a single 'T' base overhang at the 3’ end. After adapter ligation, DNA fragments were size-selected using AmpureXP beads to remove excess adapters and adapter-modified DNA fragments were enriched by PCR amplification with Illumina primers for 12 cycles. The purified ChIP-seq libraries thus generated were validated and subsequently captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Illumina HiSeq2500/ HiSeq4000 system following the manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
BWA (Li, 2010 #9905;Li, 2009 #9908) or BOWTIE2 (Langmead, 2012 #9901) was used to map ChIP-seq reads onto the S. pombe genome assembly EF2 (ASM294v2) or modified genome assembly generated based on ASM294v2, where centromere 2 was replaced with sequence from centromere 1 to mimic (cc2∆::cc1) and to include pHCC2 plasmid sequence.
Indexed, sorted bam files were created for each dataset using SAMtools (Li, 2009 #9907).
The program bamCoverage from the deepTools suite version 2.5.3 (Ramirez, 2016 #9919) or BEDtools (Quinlan, 2014 #9916;Quinlan, 2010 #9917) was used to create bigwig files from the mapped bam files and scaled using reads per kilobase of transcript per million mapped reads [RPKM]
SeqPlots (Stempor, 2016 #9923) was used to plot the occupancy over transcription start sites [TSS] as annotated in PomBase.
Genome_build: S. pombe EF2 genome assembly; ASM294v2
Genome_build: S. pombe +pHCC2 genome (see pombe.ASM294v2.20.cc2_D.gtf.gz, pombe_cc2D_pHcc2.bed.gz, pombe_cc2_D_pHetcc2.ann.gz, pombe_cc2_D_pHetcc2.fa.gz)
Supplementary_files_format_and_content: bigWig
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| Submission date |
Nov 06, 2017 |
| Last update date |
Dec 01, 2020 |
| Contact name |
Alastair Robert Kerr |
| E-mail(s) |
alastair.kerr@ed.ac.uk
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| Organization name |
University of Edinburgh
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| Department |
Wellcome Centre for Cell Biology
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| Lab |
Bioinformatics Core Facility
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| Street address |
2.21 Michael Swann Building, Kings Buildings, Mayfield Road
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| City |
Edinburgh |
| State/province |
Midlothian |
| ZIP/Postal code |
EH9 3JR |
| Country |
United Kingdom |
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| Platform ID |
GPL22682 |
| Series (1) |
| GSE106585 |
Swr1 mediated H2A.ZPht1 incorporation designates centromere DNA for de novo CENP-ACnp1 assembly |
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| Relations |
| BioSample |
SAMN07982112 |
| SRA |
SRX3367839 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2843470_Pht1-myc_swr1D_Rep3.bw |
45.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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