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Sample GSM3754746 Query DataSets for GSM3754746
Status Public on Sep 20, 2019
Title pTATA-loz1GFP_MinusZn_input1
Sample type SRA
 
Source name cells
Organism Schizosaccharomyces pombe
Characteristics genotype: pgk1DTATA-Loz1GFP
chip antibody: none
zinc supplementation: no
Growth protocol 75 ml of the strain loz1Δ pgk1(ΔTATA)-Loz1GFP were grown to an OD600 of ~6.0 in ZL-EMM supplemented with or without 100 mM Zn2+ and were treated with 1% formaldehyde for 10 min at room temperature. Crosslinking was quenched by the addition of 250 mM glycine.
Extracted molecule genomic DNA
Extraction protocol Cells were then washed, resuspended in the lysis buffer (50mM Hepes-KOH pH 7.5, 140mM NaCl, 1mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) sodium deoxycholate, 0.4mM DTT, and protease inhibitors) and lysed by vortexing for 7 x 1 min in the presence of zirconium beads. To obtain cross-linked chromatin, cell lysates were centrifuged at 14,000 rpm for 5 min and the supernatant discarded. After centrifugation, cross-linked chromatin was resuspended in lysis buffer, DNA sheared to an average length of 500 bp by sonication using Covaris Evolution, and cell debris removed by centrifugation at 14000 rpm for 5 min. For immunoprecipitations, chromatin solutions were incubated with anti-GFP antibodies (Abcam Ab290) and protein A magnetic beads (Invitrogen™ Dynabeads™ Protein A) overnight at 4°C. After Immuno-complexes were harvested the crosslinks were reversed by heating at 65°C for 5-7 hrs.
The ChIP-seq library preparation and sequencing were performed by the Institute for Genomic Medicine at Nationwide Children’s Hospital. ChIP-seq libraries were constructed from 5 ng of fragmented ChIP DNA using NEB Ultra II FS library prep kit (New England Biolabs, Ipswhich MA). Briefly, DNA fragment ends were 5´ Phosphorylated, dA-tailed, and ligated with a unique, dual UMI indexed adaptor (Integrated DNA Technologies, Iowa). Following purification, using a magnetic-bead based approach (AMPure XP System), adaptor-ligated DNA was amplified by 8-9 PCR cycles. Quality of libraries were determined via Agilent 2200 TapeStation using High Sensitivity D1000 reagents, and quantified using Kappa SYBR®Fast qPCR kit (KAPA Biosystems, Inc, MA).
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina MiniSeq
 
Data processing Sequencing reads were analyzed using the HOMER suite (Heinz et al., 2010).
Tag directories were created using HOMER makeTagDirectory with -fragLength parameter set for each sample.
UCSC bedgraph files were generated for each sample using HOMER makeUCSCfile
Genome_build: Schizosaccharomyces_pombe.ASM294v2
Supplementary_files_format_and_content: UCSC bedgraph files generated using HOMER makeUCSCfile
 
Submission date May 07, 2019
Last update date Sep 20, 2019
Contact name Stevin Wilson
E-mail(s) wilson.3273@osu.edu
Organization name The Ohio State University
Department Molecular Genetics
Street address 1787 Neil Ave, 063
City Columbus
State/province Ohio
ZIP/Postal code 43210
Country USA
 
Platform ID GPL25575
Series (2)
GSE130820 The zinc-responsive repressor Loz1 is the master regulator of zinc homeostasis in Schizosaccharomyces pombe [ChIP-seq]
GSE130846 The zinc-responsive repressor Loz1 is the master regulator of zinc homeostasis in Schizosaccharomyces pombe
Relations
BioSample SAMN11586949
SRA SRX5802035

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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