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Sample GSM3669489 Query DataSets for GSM3669489
Status Public on Jun 18, 2019
Title GS216 MM6 H3K27me3 ChIPseq rep1
Sample type SRA
 
Source name AML cell line MM6
Organism Homo sapiens
Characteristics chip antibody: H3K27me3 (Active Motif, 39161, lot 21518003)
cell line: MM6
cell type: Acute myeloid leukemia (AML)
Treatment protocol no treatment
Growth protocol cells were cultured following the recommendations of the dsmz; medium composition: 90% RPMI 1640 + 10% h.i. FBS + 2 mM L-glutamine + 1x non-essential amino acids + 1 mM sodium pyruvate for MM1 and 90% RPMI 1640 + 10% h.i. FBS + 2 mM L-glutamine + non-essential amino acids + 1 mM sodium pyruvate + 10 µg/ml human insulin for MM6
Extracted molecule genomic DNA
Extraction protocol formaldehyde-fixed cells (1% formaldehyde, 10min RT) were lysed in Buffer B (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0,3%SDS, 1x protease inhibitors -Roche) and sonicated in a microTUBE on a Covaris S220 until most of the DNA fragments were 200-500 base pairs long (settings: duty cycle 2%, peak incident power 105 Watts, cycles per burst 200, 25 min). After shearing lysates were centrifuged (10min 4°C 12000g) and supernatant diluted with 1 volume of Dilution Buffer (1mM EGTA 300 mM NaCl, 2% Triton x-100, 0.2% DOC, 1x protease inhibitors-Roche). In parallel, Dynabeads (Protein A) were conjugated to the antibody (2ul, H3K27ac, diagenode, cat no: C15410174, lot no: A.7071-001P; 1.5ul, H3K27me3, Active Motif, cat no: 39161, lot no: 21518003) in PCR tubes by rotating 1.5 hour at 4°C (30rpm). Antibody-conjugated magnetic beads were added to the tube containing the chromatin and incubated 3h at 4°C (30rpm). Beads were washed (20rpm; 10min) four times with 500 ul Buffer A (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM EGTA,1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl, 1x protease inhibitors) and once with 500 ul Buffer C (10 mM Tris-HCl, pH 8.0, 10 mM EDTA). Beads were then incubated with 70 μl elution buffer (0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris HCl pH 8.0) containing 2μl of RNase (10mg/ml) for 30 min at 37°C, 900 rpm, then 1h with 2 μl of Proteinase K (20mg/ml) at 55°C, 900 rpm, and finally overnitht at 65°C to revert formaldehyde crosslinking; beads were magnetized and supernatant was transferred to a new tube. Another 30 μl of elution buffer + 1μl of proteinase K were added to the beads and the tubes were incubated 1h with the same conditions than before; the eluates were combined. Finally DNA was purified with AMPure XP beads.
Ultra II DNA Library prep kit for Illumina (NEB E7645S) was used for library preparation
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 1500
 
Data processing ChIP-seq reads were aligned to the human genome (hg38) using bowtie (Langmead et al., 2009) with options “-q -n 2 --best --chunkmbs 2000 -p 32 -m1”
Genome_build: hg38
Supplementary_files_format_and_content: bigWig files were generated from homer tag directories using makeBigWig.pl
 
Submission date Mar 13, 2019
Last update date Jun 18, 2019
Contact name Helena Domínguez Moreno
Organization name Biomedical Center of LMU
Department Molecular Biology
Lab AG Schotta
Street address Großhaderner Str. 9
City Planegg
State/province Bavaria
ZIP/Postal code 82152
Country Germany
 
Platform ID GPL18460
Series (2)
GSE128259 Loss of DKM6A confers drug resistance in acute myeloid leukemia (ChIP-seq of AML cell lines MM-1 and MM-6)
GSE128262 Loss of DKM6A confers drug resistance in acute myeloid leukemia
Relations
BioSample SAMN11119520
SRA SRX5516741

Supplementary file Size Download File type/resource
GSM3669489_GS216_MM6_H3K27me3_ChIPseq_m1.ucsc.bigWig 131.4 Mb (ftp)(http) BIGWIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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