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Sample GSM2412282 Query DataSets for GSM2412282
Status Public on Mar 27, 2018
Title MYB DMSO vehicle
Sample type SRA
 
Source name THP1 AML cells
Organism Homo sapiens
Characteristics cell type: AML
Treatment protocol No additional treatment
Growth protocol THP1 cells were cultured in RPMI 1640 without supplemental growth factors.
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).
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
 
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.
 
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
Organization name Cancer Research UK Manchester Institute
Lab Leukaemia Biology Laboratory
Street address Wilmslow Road
City Manchester
State/province Lancashire
ZIP/Postal code M20 4BX
Country United Kingdom
 
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]
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 SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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