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| Status |
Public on Sep 03, 2013 |
| Title |
CD34_SCL_ChIP-seq |
| Sample type |
SRA |
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| Source name |
CD34+ cells
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| Organism |
Homo sapiens |
| Characteristics |
chip antibody: SCL (Santa Cruz, sc-12984X), B2312
cell type: CD34+ cells
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Bone marrow (BM) samples were harvested from normal volunteers with informed consent in accordance with local ethical guidelines. The CD34+ fraction was obtained by magnetic bead separation using an automated CliniMACS cell separation system (Miltenyi Biotec, Cologne). Cell purity was assessed by flow cytometry and was ≥ 98%. ChIP assays were performed with 15 x 10^6 per condition. Total RNA extraction and purification was performed using miRNeasy mini kits (QIAGEN). The small RNA fraction (<200nt) was collected in a separate fraction from total RNA, as per manufacturer’s standard instructions. Total RNA was amplified using the Ovation RNA-seq system V2 (NuGEN) prior to sequencing.
For ChIP, cells were harvested and washed with phosphate-buffered saline (PBS), followed by incubation of 1% (w/v) formaldehyde for 10 minutes at room temperature. To terminate the cross-link, cells were incubated with 0.125M glycine for 5 minutes. Cells were washed with PBS and lysed on ice in Cell lysis buffer (10mM Tris [pH 8.0], 10mM NaCl, 0.2% NP-40) for 10 minutes to recover nuclei. After centrifugation at 1500xg for 5 minutes, nuclei were lysed in Nucleus lysis buffer (50 mM Tris, 10mM EDTA, 1% SDS [pH 8.0]) on ice for 10 minutes. The lysate was diluted in IP dilution buffer (20mM Tris [pH 8.0], 2mM EDTA, 150mM NaCl, 1% Triton-X100, 0.01% SDS) and sonicated (Settings: High, 30sec pulses) using BioRuptor® sonicator (Diagenode, Liège, Belgium) to yield an average fragmentation size of approximately 200bp. The chromatin was pre-cleared with 100?g rabbit IgG for 1 hour followed by incubation with 100?l protein-G-agarose (Roche Applied Science, Penzberg, Germany) for 2 hours. For each sample, 300?l pre-cleared chromatin was removed (input for the subsequent qRT-PCR analysis) and the remaining chromatin was aliquoted, and incubated with the indicated antibody for 18 hours at 4°C. To collect immune complexes, 50?l protein G-agarose was added to the chromatin and incubated for additional 2 hours at 4°C. Protein-G-agarose pellets were washed at 5000xg; twice with 500?l IP wash buffer 1 (20mM Tris [pH 8.0], 2mM EDTAm 50mM NaCl, 1% Triton-X100, 0.1% SDS), once with IP wash buffer 2 (10mM Tris [pH 8.0], 1mM EDTA, 0.25M LiCl, 1% NP-40, 1% Sodium deoxycholate) and twice with TE (10mM Tris, 1mM EDTA [pH8.0]). Immuno-precipitated chromatin was eluted in 300?l Elution Buffer (100mM NaHCO3, 1% SDS) and reverse-cross link was obtained by incubation with RNase A and NaCl (0.3M final concentration) at 67°C for 18 hours followed by treatment with Proteinase K at 45°C for 2 hours. Input DNA (pre-cleared chromatin) was treated with RNase A and Proteinase K simultaneously.
Library construction was performed as per Illumina protocol Rev A 11257047
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Base-calling: Illumina Real Time Analysis, scoring metric v1.5
Raw reads were prefiltered to remove all reads that have 3 or more bases with a quality score of less than Q13
All provided cleaned data have had adaptors clipped except the small RNA-seq data which still contains 5' adaptor: 5'-TGGAATTCTCGTATGCCGTCTTCTGCTTG
Alignments were all made against hg19. The following aligners were used. ChIP-seq: bwa (version 0.6.1-r104) default parameters , mRNA-seq: tophat (version 1.3.1) -g 25 -r 20 --segment-length 45 -G "hg19_refseq.gtf" --solexa1.3-quals, smallRNA: bowtie (version 0.12.5) -v 2 -m 25 --solexa1.3-quals --best --strata.
Bed files generated from BAM files using bedtools bamtobed (version 2.15.0). For ChIP-seq files, each bed entry is extended to 200bp in length. For RNA-seq the split option was used.
Genome_build: hg19
Supplementary_files_format_and_content: Bed files contain coordinates for all aligned reads
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| Submission date |
Mar 13, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Jason Wong |
| E-mail(s) |
jason.wong@unsw.edu.au
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| Organization name |
UNIVERSITY OF NEW SOUTH WALES
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| Street address |
Level 2, Lowy Cancer Research Centre, University of New South Wales
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| City |
Sydney |
| State/province |
NSW |
| ZIP/Postal code |
2052 |
| Country |
Australia |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE45144 |
Genome-wide Analysis of Transcriptional Regulators in Human Blood Stem/Progenitor Cells reveals a densely interconnected network of coding and non-coding genes. |
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| Relations |
| SRA |
SRX248932 |
| BioSample |
SAMN01975654 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1097881_CD34_SCL_ChIP-seq.bed.gz |
337.8 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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