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| Status |
Public on Jun 30, 2024 |
| Title |
ChIP-seq-H3K4me3_NB1-M11_TUM |
| Sample type |
SRA |
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| Source name |
Tumor cells
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| Organism |
Homo sapiens |
| Characteristics |
patient: NB1
pdxs: M11
cell type: Tumor cells
chip antibody: H3K4me3 (Millipore 07-473)
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| Growth protocol |
The tumor samples were subjected to mechanical and enzymatic dissociation using Tumor Dissociation Kit (Miltenyi Biotec). The dissociated tumor cells were suspended in DMEM/F12 medium supplemented with 1% penicillin-streptomycin, 2% B27 (Invitrogen), and 20ng/ml of human recombinant FGF and EGF (Peprotech). Adherent cells were cultured in DMEM (catalog no. Gibco) supplemented with 10% of FBS (PAN-Biotech) and 1% of penicillin-streptomycin (Gibco). Cells were maintained at 37°C in humidified, 5% CO2 chambers. Spheroid was grown in ultralow attachment plates (Corning).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Single cells from adherent or spheroids cultures counted and diluted to a target of 500’000 cells per ml. Formaldehyde at 1% final concentration was added for 10 minutes at 37°C. Quenching was performed with 125 mM Glycine. The solution was spun at 1500rpm for 5’ at 4C and pellet washed twice with cold PBS + protease inhibitors (PI) before snap freezing. For tumors ~40mg a fragment was put in petri dish on normal ice and cut into small pieces and resuspended in 1ml PBS + protease inhibitors and transferred to a 1.5ml tube. Formaldehyde at 1% final concentration was added for 10 minutes at 37°C. Quenching was performed with 125 mM Glycine. Pellet was washed once in cold PBS + PI after 5min spin at 2,500 RPM at 4°C. Manual dissociation in PBS + PI was performed with syringe passages: 10up/down with a 18G needle, 10up/down with a 21G needle, 10up/down with a 23G needle (if possible). The solution was spun down another time and the pellet snap frozen. Lysis was performed at 4°C for 10min in 300ul of 50mM Tris-HCl, 1% SDS, 0.25% DOC (+ PI) and sonication was performed with tip sonicator after dilution in 900ul of dilution buffer (DB) - 50mM Tris-HCl pH 7.4, 0.1% SDS, 150mM NaCl, 1.84% Triton-X + PI - with the following parameters: sonication time, 3 min; amplitude, 40%; cycle, 0.7s ON and 1.3s OFF. After clearing the solution with 14000rpm spin, the supernatant was further diluted with 1,8ml of DB before IP overnight at 4°C. The equivalent of 2-5M cells or 5-10mg of tumors was used for a single immunoprecipitation (IP) with 0.1-1ug antibody. After washing once Protein G magnetic beads in DB, the equivalent of 30 ul (50 ul for tumors) of original solution was added to the IPs for 2h at 4°C. Beads were washed 2x with 150 mM NaCl RIPA buffer, 500mM NaCl RIPA, LiCl buffer (10mM Tris-HCl pH 8.1, 250mM LiCl, 0.5% Triton X-100, 0.5% DOC) and once in 10 mM Tris-Cl pH 8.5. Elution was performed with 1X TE pH 8.0, 0.1% SDS, 150mM NaCl, 5mM DTT at 65°C for 1h. RNAse treatment and crosslink reversal with proteinase K were applied on the supernatant, before SPRI beads DNA purification.
After Qubit quantification 5-10ng DNA was used for Illumina TruSeq Library preparation. Sequencing was performed on a Hi-Seq Illumina Genome Analyzer (50 bp single-read).
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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 |
ChIP-seq reads were trimmed using trim_galore (version 0.6.4 options -q 20 –stringency 2)
Reads were mapped to the hg38 genome (only chromosomes 1 to 22, X, Y and M) using bowtie2 (version 2.3.0) and only reads with mapping quality >= 10 were kept.
Read density tracks were generated using bedtools genomecov (version 2.29.0) for each genomic position from reads extended to 200bp and read counts were normalized to one million reads in the library and visualized using the IGV browser.
Peaks were called using MACS2 (version 2.2.5 option --broad-cutoff 0.00001) and peaks located on chromosome M or in ENCODE blacklisted regions were removed.
For comparison across samples, ChIP-seq peak regions were merged using bedtools merge (version 2.29.0) and raw read counts per region were calculated using bedtools intersect (version 2.29.0 option -c). Differentially enriched regions were identified using DESeq2 (version 1.26.0) using the following thresholds: adjusted p-value <= 0.05 and fold change >= 2.
Assembly: hg38
Supplementary files format and content: bigWig
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| Submission date |
Jan 27, 2023 |
| Last update date |
Jun 30, 2024 |
| Contact name |
Anais Flore Bardet |
| Organization name |
IGBMC
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| Street address |
1 rue Laurent Fries
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| City |
Illkirch |
| ZIP/Postal code |
67404 |
| Country |
France |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE223901 |
Pre-clinical spheroid models identify BMX as a therapeutic target for metastatic MYCN non-amplified neuroblastoma (ChIP-Seq) |
| GSE223902 |
Pre-clinical spheroid models identify BMX as a therapeutic target for metastatic MYCN non-amplified neuroblastoma |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM6998526_ChIP-seq-H3K4me3_NB1-M11_TUM.bw |
125.5 Mb |
(ftp)(http) |
BW |
| Raw data not provided for this record |
| Processed data provided as supplementary file |
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