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Sample GSM1373641 Query DataSets for GSM1373641
Status Public on Jul 10, 2014
Title nts1_chip
Sample type SRA
 
Source name yeast cells
Organism Schizosaccharomyces pombe
Characteristics strain: NB4385
genotype: leu1-32 ura4-D18 hphMX6:tetO7-TATACYC1-FLAG3-nts1-TAP:kanMX6
chip antibody: IgG (GE Healthcare, catalog# 17-0969-01, for protein A epitope) & anti-FLAG (M9, Sigma, catalog# M8823, for FLAG epitope)
Growth protocol Cells were grown at 30 °C in YES to mid-logarithmic phase
Extracted molecule genomic DNA
Extraction protocol 700 OD600 of cells were fixed in 1% w/v formaldehyde for 25 min at room temperature. The reaction was stopped by adding glycine to a final concentration of 125 mM. After extensively washing the pellets in Tris-buffered saline solution, the cells were aliquoted into 14 equal samples. Cells were lysed by bead beating in 0.5 mL per sample of 50 mM HEPES pH 7.6, 140 mM NaCl, 1 mM EDTA, 0.1% v/v Triton-X 100, 1 mM PMSF, 1× Complete Protease Inhibitor Cocktail. Following addition of Triton X-100 to 1% v/v, chromatin was sheared by sonication in a Misonix Sonicator 3000 (Farmingdale, NY, 15 cycles of 30 s ON at maximum intensity and 1 min OFF). The lysate was cleared by centrifugation and adjusted to 10 mL at 15 mg mL-1 of total protein, as determined by the Bradford assay. This lysate was incubated with 0.5 mL of pre-equilibrated IgG resin (GE Healthcare Life Sciences) overnight at 4 °C with rotation. Next, the resin was washed three times with 1 mL of the lysis solution described above without protease inhibitors, once with 50% the same solution and 50% of 20 mM HEPES pH 7.6, 150 mM NaCl, 0.1% v/v NP-40, 1 mM EDTA and once with 100% of the latter solution. The IgG resin was resuspended with 0.75 mL of the latter solution supplemented with 0.75 mM DTT and 500 U of TEV protease (Eton Biosciences, San Diego, CA) and incubated at room temperature for 4 h with rotation. The resin was washed three times with 0.5 mL of the same solution used for proteolysis, which were combined with the eluate, supplemented with 0.2 mL of pre-equilibrated anti-FLAG magnetic resin (M2, Sigma-Aldrich), and incubated overnight at 4 °C with rotation. The anti-FLAG resin was washed three times for 10 min with 10 mL of 50 mM HEPES pH 7.6, 140 mM NaCl, 1 mM EDTA, 1 % v/v Triton-X 100, 0.1% v/v sodium deoxycholate, twice more in the same buffer but containing 500 mM NaCL, twice in 10 mM Tris-HCl pH 8, 250 mM LiCl, 1 mM EDTA, 0.5% v/v NP-40, 0.5% v/v sodium deoxycholate and once in Tris-EDTA solution. The resin was eluted twice with 0.5 mL of 10 mM Tris pH 8, 1 mM EDTA, 1% v/v SDS for 15 min at 70 °C. The eluted DNA was de-crosslinked overnight at 65 °C, purified with Qiagen’s PCR Purification Kit (Qiagen, Germantown, MD), and eluted in 50 µL of TE.
Libraries were prepared from 10 ng of DNA using the NEBNext® Ultra™ DNA Library Prep Kit for Illumina (NEB) following manufacturer’s recommended protocol. The libraries were amplified with 18 cycles of PCR and size selected on a 2% agarose gel to remove adapter dimer products. The purified DNA library was denatured in 0.1 N NaOH and diluted to a final concentration of 10 pM before being loaded onto an Illumina single read flow-cell.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2000
 
Description Sample 2
Data processing fastx_trimmer -f 7 -l 47 -Q33 (trim first 6 and last 47 nt of each read)
fastq_quality_filter -q 28 -p 95 -Q33 (filter - 95% of bases quality score >28)
bowtie -q -p 8 -S -n 2 -e 70 -l 40 --maxbts 800 -y -k 1--best --phred33-quals (align to reference genome and output SAM file, reads that did not align were not included in the output SAM file)
picard SortSam SORT_ORDER=coordinate (sort SAM file)
picard MarkDuplicates (mark PCR duplicates, input = 141599 reads, chip = 360672 reads)
picard DownsampleSam PROBABILITY=0.4 RANDOM_SEED=1 (downsample ChIP alignment file to 40%)
macs --gsize=12462637 --bw 600 -w (peak calling)
Genome_build: ASM294v2.22 (ftp://ftp.ensemblgenomes.org/pub/fungi/current/fasta/schizosaccharomyces_pombe/dna/)
Supplementary_files_format_and_content: bed and wig files were generated using MACS
 
Submission date Apr 24, 2014
Last update date May 15, 2019
Contact name Nicola Zilio
Organization name The Scripps Research Institute
Street address 10550 N Torrey Pines Road
City San Diego
State/province CA
ZIP/Postal code 92037
Country USA
 
Platform ID GPL13988
Series (1)
GSE57040 A Novel HDAC Complex in the Control of Transcription and Genome Stability
Relations
BioSample SAMN02736805
SRA SRX525989

Supplementary file Size Download File type/resource
GSM1373641_nts1_chip.wig.gz 2.4 Mb (ftp)(http) WIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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