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| Status |
Public on May 31, 2019 |
| Title |
CBX8-untreated |
| Sample type |
SRA |
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| Source name |
K562
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| Organism |
Homo sapiens |
| Characteristics |
antibody: anti-CBX8 (C15410333, Diagenode)
treatment: untreated
sample collection (day): 1
cell line: K562
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| Treatment protocol |
Cells were heat shocked for 1 hour at 44C
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| Growth protocol |
The (human) erythromyeloblastoid leukemia cell line K562 was cultured in RPMI 1640 (containing L-glutamine) supplemented with 10% FCS and 1% penicillin/streptomycin (PAA Laboratories).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Control and HS K562 cells were crosslinked with 1% formaldehyde for 10 minutes at room temperature at a concentration of 15x106 cells/ml. The fixed cells were sonicated for 4 minutes (30 sec on; 30 sec off) using the Diagnode Bioruptor and centrifuged at maximum speed for 10 minutes. Chromatin of about 5x105 cells was incubated overnight in dilution buffer (167mM NaCl, 16,7 mM Tris (pH 8), 1,2mM EDTA, 1% Triton X-100) with 1 µg antibody at 4C. ProtA/G beads were blocked and incubated with the chromatin-Ab for one hour at 4C. Beads were washed with three different wash buffers and chromatin was eluted from the beads. DNA-proteins were de-crosslinked (200mM NaCl and 4 µl proteinase K (10mg/ml)), by incubation for four hours at 65C and samples were purified using the Qiaquick MinElute PCR purification kit according manufacturer’s protocol.
Sequencing samples were prepared according to the manufacturer's protocol (Illumina). End repair was performed using the precipitated DNA using Klenow and T4 PNK. A 3’ protruding A base was generated using Taq polymerase and adapters were ligated. The DNA was loaded on gel and a band corresponding to ~300 bp (ChIP fragment + adapters) was excised. The DNA was isolated, amplified by PCR and used for cluster generation on the NextSeq500 genome analyzer. The 50 bp tags were mapped to the human genome HG19 using BWA (Li and Durbin, 2009). For processing and manipulation of SAM/BAM files SAMtools was used (Li et al., 2009). For each base pair in the genome the number of overlapping sequence reads was determined and averaged over a 10 bp window and visualized in the UCSC genome browser (Kent et al., 2002).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Sequencing reads were aligned to the human genome hg19 using bwa and bam2bw was used for visualization.
Genome_build: hg19
Supplementary_files_format_and_content: bigwig (bw) files
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| Submission date |
Oct 12, 2018 |
| Last update date |
Nov 11, 2021 |
| Contact name |
Joost Martens |
| E-mail(s) |
j.martens@science.ru.nl
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| Phone |
0243780645
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| Organization name |
Radboud University
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| Department |
RIMLS
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| Lab |
Molecular Biology
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| Street address |
Geert Grooteplein 28
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| City |
Nijmegen |
| State/province |
Nederland |
| ZIP/Postal code |
6525GA |
| Country |
Netherlands |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE121182 |
Dynamic behavior of the epigenetic machinery upon thermal stress |
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| Relations |
| BioSample |
SAMN10236330 |
| SRA |
SRX4872742 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3427438_CBX8-untreated-ChIP-13690.bam.filtered.bam.bw |
57.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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