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| Status |
Public on Jun 18, 2019 |
| Title |
GS701 MM6 H3K27me3 ChIPseq rep2 |
| Sample type |
SRA |
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| Source name |
AML cell line MM6
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| Organism |
Homo sapiens |
| Characteristics |
chip antibody: H3K27me3 (Active Motif, 39161, lot 21518003)
cell line: MM6
cell type: Acute myeloid leukemia (AML)
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| Treatment protocol |
no treatment
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| 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
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| 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
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 1500 |
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| 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
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| Submission date |
Mar 13, 2019 |
| Last update date |
Jun 18, 2019 |
| Contact name |
Helena Domínguez Moreno |
| Organization name |
Biomedical Center of LMU
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| Department |
Molecular Biology
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| Lab |
AG Schotta
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| Street address |
Großhaderner Str. 9
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| City |
Planegg |
| State/province |
Bavaria |
| ZIP/Postal code |
82152 |
| Country |
Germany |
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| 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 |
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| Relations |
| BioSample |
SAMN11119519 |
| SRA |
SRX5516742 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3669490_GS701_MM6_H3K27me3_ChIPseq_m1.ucsc.bigWig |
199.0 Mb |
(ftp)(http) |
BIGWIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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