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| Status |
Public on Jun 26, 2017 |
| Title |
sKH1_Anti-RUNX1_SIREVI1_D10_ChIPseq |
| Sample type |
SRA |
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| Source name |
RUNX1-EVI1 KD SKH-1 cells after 10 days
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| Organism |
Homo sapiens |
| Characteristics |
cell type: SKH-1 cells
genotype: RUNX1-EVI1 KD after 10 days
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Each aliquot of 2x107 cells was resuspended in 600μl of sonication buffer (Tris-HCL pH8 25mM, NaCL 150mM, EDTA 2mM, Triton 100x 1%, SDS 0.25%, Protease inhibitior cocktail (PIC) 1x). 300 μl of nuclei in sonication buffer was placed in each polystyrene tube and sonicated at 75% amplitude, 26 cycles: 30s on and 30s off per cycle (Q800, Active Motif, USA). Subsequently, 1.2ml of dilution buffer (Tris-HCL pH8 25mM, NaCL 150mM, EDTA 2mM, Triton 100x 1%, glycerol 7.5%, PIC 1x) was added to the pooled post sonication material. This was divided equally between four immunoprecipitations (with 5% of input taken for validation).15 μl protein G beads (Diagenode, Belgium) were washed twice with 500 μl of 50mM citrate phosphate buffer and once with 100mM sodium phosphate). 2μg antibody (EVI-1, C50E12, Cell signalling, lot 3; or RUNX1, Ab23980, Abcam lot 144722) or 4µg antibody (C/EBPα, A2814 Santa Cruz) was added to 10 μl 100mM sodium phosphate, 0.5% BSA and incubated with protein G beads at 4°C for 1 hour. Chromatin was then added to the protein G beads with antibody and returned to 4°C for 4 hours. Unbound chromatin was separated from the beads by magnet and the attached beads were washed by buffer 1 (Tris HCL 20mM, NaCl 150mM, EDTA 2mM, Triton x100 1%, SDS 0.1%), twice with buffer 2 (Tris HCL 20mM, NaCl 500mM, EDTA 2mM, Triton x100 1%, SDS 0.1%), LiCL buffer (Tris HCL 10mM, LiCl 250mM, EDTA 1mM, NP40 0.5%, sodium deoxychlolate 0.5%) and finally twice with wash buffer 4 (Tris HCL pH8, 10mM, NaCl 50mM, EDTA 1mM). The column was eluted twice with 50 μl buffer (NaHCO3 100mM and SDS 1%) and the eluant containing the chromatin was pooled. Crosslinks were reversed by incubating the samples at 65°C overnight in 500mM NaCl, 500 μg/ml proteinase K. DNA was purified by Ampure beads (Beckman Coulter, USA), as above, with the DNA eluted with 50 μl water. Validation of the ChIP was performed by qPCR using a standard curve of genomic DNA from untreated SKH-1 cells (10ng/ μl followed by serial 1:5 dilutions). The input material was diluted 1:5 with water and qPCR was performed as above with primers listed in SI. Validation was analysed as a ratio of the qPCR signal from the ChIP material over the input.
Libraries for high throughput sequencing were prepared using the Tru-seq DNA sample preparation kit (Illumina, USA) or Kapa HyperPrep kit (Kapa biosystems, USA), as per manufacturer’s protocol. 18 cycles of PCR was performed and 200-350bp fragments were size selected by running the samples in an agarose gel. Libraries were purified from the gel using a Minieluta Gel extraction kit (Qiagen, USA). Libraries were validated by qPCR, with an analysis of the ChIP signal of a positive control region (e.g. PU.1 3H enhancer) over a negative control region (e.g. IVL). Finally, libraries were quantified by Kapa library quantification kit (Kapa biosystems, USA) and run in a pool of four indexed libraries in one lane of a HiSeq 2500 (Illumina, USA) or 12 indexed libraries in one lane of a NextSeq 500 (Illumina, USA) using 50 cycle single-end reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Quality of reads was verified using FASTQC analysis
Sequences reads in fastq format were mapped onto the reference human genome version hg38, Genome Reference Consortium GRCh38. The Illumina reads were aligned to the human genome using Bowtie2
Reads that were uniquely aligned to chromosomal positions were retained and duplicate reads were removed from the aligned data using Picard tools.
The filtered aligned reads were used to generate density profiles using “genomeCoverageBed” function from bedtools and then converted to big wig file using "wigToBigWig" function
Genome_build: hg38
Supplementary_files_format_and_content: bigWig
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| Submission date |
Sep 22, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Salam Adli Assi |
| E-mail(s) |
s.a.assi@bham.ac.uk
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| Organization name |
University of Birmingham
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| Department |
Institute for Cancer and Genomic Sciences
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| Street address |
IBR
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| City |
Birmingham |
| ZIP/Postal code |
B15 2TT |
| Country |
United Kingdom |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE87283 |
RUNX1-ETO and RUNX1-EVI-1 differentially program the chromatin landscape in t(3;21) and t(8;21) AML but share global C/EBP-alpha dysfunction (ChIP-Seq) |
| GSE87286 |
RUNX1-ETO and RUNX1-EVI-1 differentially program the chromatin landscape in t(3;21) and t(8;21) AML but share global C/EBP-alpha dysfunction |
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| Relations |
| BioSample |
SAMN05806616 |
| SRA |
SRX2187098 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2326840_sKH1_RUNX1_SIREVI1_D10_ChIPseq.bw |
142.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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