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| Status |
Public on Mar 27, 2018 |
| Title |
MYB DMSO vehicle |
| Sample type |
SRA |
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| Source name |
THP1 AML cells
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| Organism |
Homo sapiens |
| Characteristics |
cell type: AML
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| Treatment protocol |
No additional treatment
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| Growth protocol |
THP1 cells were cultured in RPMI 1640 without supplemental growth factors.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
THP1 cells were cultured for 48 hours in the presence of DMSO or OG86 250nM at a density of 3x105/ml. Cells were cross-linked at room temperature using 1% formaldehyde. After 10 minutes the reaction was stopped by incubation for 5 minutes with 0.125M glycine. Cell pellets were washed twice with cold PBS containing protease inhibitors (Complete EDTA-free tablets, Roche, Basel, Switzerland). 100 million cells were used per ChIP, as described in the protocol reported by Lee et al. (2006). Briefly, nuclear lysates were sonicated using a Bioruptor Plus (Diagenode) for 15 min at high, 30 sec ON, 30 sec OFF settings. Immunoprecipiation was performed overnight at 20 rpm and 4°C, with 100ul magnetic beads (Dynabeads (Protein G), Invitrogen, Carlsbad, CA) per 10µg antibody. ChIP-grade antibodies were used as follows: LSD1 (ab17721), GFI1 (ab21061) and MYB (ab45150) (Abcam). After washing six times with RIPA buffer (50mM HEPES pH 7.6, 1mM EDTA, 0.7% Na deoxycholate, 1% NP-40, 0.5M LiCl), chromatin IP-bound fractions were extracted at 65°C for 30min with elution buffer (50mM TrisHCl pH8, 10mM EDTA, 1% SDS) vortexing frequently. RNAseA (1mg/ml) and proteinase K (20mg/ml) were used to eliminate any RNA or protein from the samples. Finally DNA was extracted using phenol:chloroform:isoamyl alcohol extraction and precipitated with ethanol (adding 2 volumes of ice-cold 100% ethanol, glycogen (20µg/µl) and 200mM NaCl) for at least 1 hour at -80°C. Pellets were washed with 70% ethanol and eluted in 50ul 10mM TrisHCl pH8.0.
ChIP DNA samples were prepared for sequencing using the Microplex Library Preparation Kit (Diagenode) and 1 ng ChIP DNA. Libraries were size selected with AMPure beads (Beckman Coulter) for 200-800 base pair size range and quantified by Q-PCR using Kapa Library Quantification Kit (Kapa Biosystems).
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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 |
Reads were aligned to human genome hg38 using Bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/) using default settings. The number of uniquely mapped reads per sample was 50-100 million. Reads were mapped relative to annotated genes (ENSEMBL v66) using the Annmap database, R and Bioconductor (Gentleman et al., 2004; Yates et al., 2007).
MACS2 (Model-based Analysis of ChIP-seq, version 2.1.0) software was used to call peaks (Zhang et al., 2008). DMSO or OG86 treated input samples were respectively used as reference, duplicates at the exact same location were removed and a cutoff of 0.01 False Discovery Rarte (FDR) was used as a threshold. Only those peaks showing pileup values ≥18 and log(qvalue) ≥3 were considered for further analysis.
Using ChIPseeker package (R, Bioconductor) (Yu et al., 2015) peak coordinates were annotated to the nearest genomic features using transcript-related features from UCSC hg38. Transcript start site (TSS) region was defined as ±1kb from the TSS. As some peaks overlap multiple genomic regions, the package adopted the following priority in annotation: promoter, 5’ UTR, 3’ UTR, exon, intron, downstream, intergenic.
A ChIPpeakAnno package (R/Bioconductor) (Yu et al., 2015) was used to find peaks with apices located within 500 bps of one another, and to extract Fastq sequences around the summit of each peak. For motif analysis a window of ±500bp around summit was analyzed using the MEME-ChIP (Machanick & Bailey, 2011) with default parameters.
Genome_build: hg38
Supplementary_files_format_and_content: Processed data files 1-6: Binding peaks for LSD1, MYB or GFI1 in the presence or absence of OG86.
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| Submission date |
Dec 01, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Tim C Somervaille |
| E-mail(s) |
tim.somervaille@cruk.manchester.ac.uk
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| Organization name |
Cancer Research UK Manchester Institute
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| Lab |
Leukaemia Biology Laboratory
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| Street address |
Wilmslow Road
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| City |
Manchester |
| State/province |
Lancashire |
| ZIP/Postal code |
M20 4BX |
| Country |
United Kingdom |
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| Platform ID |
GPL18573 |
| Series (2) |
| GSE63222 |
Inhibitors of LSD1 target demethylase-independent activity to induce differentiation in acute myeloid leukemia |
| GSE90769 |
Inhibitors of LSD1 target demethylase-independent activity to induce differentiation in acute myeloid leukemia [anti-LSD1, MYB, and GFI1 ChIP-Seq] |
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| Relations |
| BioSample |
SAMN06094246 |
| SRA |
SRX2388745 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2412282_MYB_DMSO_bindingpeaks.txt.gz |
1.3 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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