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| Status |
Public on Oct 07, 2022 |
| Title |
MDA-MB-231 sgMLL3, H3K4me3, biological replicate2 |
| Sample type |
SRA |
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| Source name |
MDA-MB-231
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MDA-MB-231
cell type: Breast cancer cell line
chip antibody: H3K4me3 (clone C42D8, Cell Signaling Technology, 9751s, 2 μg per sample)
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| Treatment protocol |
No treatment
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| Growth protocol |
Cells were cultured in DMEM medium with 10%FBS.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Around 2 x 107 cells and two biological repeats per genotype were used for ChIP-seq experiment. Crosslinking was performed with 1% formaldehyde (Sigma, F1635) at 37°C for 10 min. A final concentration of 125 mM glycine was added for 5 minutes at room temperature to quench formaldehyde crosslinking. Cells were washed with cold PBS and harvested on ice. Cells were lysed with 1ml of SDS lysis buffer (1% SDS, 10mM EDTA, 50mM Tris-HCl pH8) in the presence of protease inhibitor (Roche, cat#11836170001) and phosphatase inhibitor (Thermo, cat#78427). Cells were sonicated using the Branson Sonifier 250 (Branson Ultrasonics 101063588) with a 20% amplitude setting for 5 min 30 s with 10 s of on and off intervals. After sonication, 1 ml of ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl pH8, 167 mM NaCl) with proteinase and phosphatase inhibitor was added to prevent SDS precipitation. After centrifugation at 14,000 rpm for 10 min at 4°C, 2 ml of supernatant were transferred to a 15 ml falcon tube and diluted with 3 ml of ChIP dilution buffer containing proteinase and phosphatase inhibitor. 50 μl of Dynabeads (Thermo Fisher Scientific 10009D), which were previously blocked with 1% BSA, were added to samples and incubated at 4°C with rotation for 1 hour. After pre-clearing, Dynabeads beads were removed. The supernatant was transferred to new 15 ml falcon tubes and 100 μl out of 5 ml volume was collected as 2% input. Antibodies were added to the pre-cleared samples for overnight incubation at 4°C with rotation. 200 μl of Dynabeads that were previously blocked with 1% BSA, were added to the sample and incubated for 6 hours at 4°C with rotation. After incubation, beads were collected and washed at 4°C with rotation following the procedure: 5 min with low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH8, 150 mM NaCl), twice 5 min with high salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH8, 500 mM NaCl) and twice 5 min with TE buffer (10 mM Tris-HCl pH8, 1 mM EDTA). After washing, beads were resuspended in 250 μl of elution buffer (1% SDS, 0.1 M NaHCO3) and incubated at 60°C for 30 min using a thermomixer (850 rpm). The supernatant was transferred to new tubes and 5 M NaCl was added for overnight de-crosslinking at 65°C. 10 μl of 0.5 M EDTA, 20 μl of 1 M Tris-HCl (pH 6.5) and 1 μl of proteinase K (20 mg/ml, NEB, P8107S) were added and incubated at 45°C for 1 hour for de-crosslinking. ChIP-DNA was isolated by using the QIAquick PCR purification kit (QIAGEN, 28104).
Libraries were prepared using the NEBNext® Ultra II DNA Library Prep Kit (NEB, E7103S) and NEBNext® Multiplex Oligos for Illumina® (Index Primers Set 1; NEB, E7335S). Samples were pooled and submitted to MSKCC Integrated Genomics Operation core for sequencing. Libraries were run over one lane of a HiSeq 4000 in a 50bp/50bp paired end run, using the HiSeq 3000/4000 SBS Kit (Illumina).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Basecalls were performed using bcl2fastq.
The raw read were quality assessed using FastQC v0.11.9, then quality trimmed using Trimmomatic 0.40 with illumina adaptor file and default values.
The surviving paired reads post quality trimming were then aligned against the human reference genome (GRCh38) with bwa-mem(arXiv:1303.3997v2), then sorted and indexed with samtools.The resulting BAM alignments were then processed through PICARD (https://github.com/broadinstitute/picard) tools for ‘MarkDuplicates’.
BigWig files were generated from the Markduplicated bam files using bamCoverage with RPKM normalization. The resulting BigWig files were then passed to computeMatrix, which generates a matrix of scores per genomic region for visualization.
Peaks were called using MACS2 with a p value of 1e-3. Super-enhancers were identified using ROSE based on H3K27ac ChIP-seq signals. The peak annotation was also taken from ROSE output.
Assembly: GRCh38/hg38
Supplementary files format and content: bigWig
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| Submission date |
Jun 22, 2022 |
| Last update date |
Oct 08, 2022 |
| Contact name |
Jihong Cui |
| E-mail(s) |
jihong.cui@einsteinmed.edu
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| Phone |
7186781277
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| Organization name |
Albert einstein college of medicine
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| Lab |
Wenjun Guo
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| Street address |
1301 Morris Park Avenue, Room 108/114
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| City |
Bronx |
| State/province |
NY |
| ZIP/Postal code |
10461 |
| Country |
USA |
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| Platform ID |
GPL20301 |
| Series (2) |
| GSE171447 |
MLL3 loss drives metastasis and therapeutic resistance by promoting a hybrid EMT state |
| GSE206656 |
MLL3 Loss Drives Metastasis by Promoting a Hybrid Epithelial-Mesenchymal Transition State [ChIP-seq histone marker] |
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| Relations |
| BioSample |
SAMN29248184 |
| SRA |
SRX15826261 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6260184_H3K4ME3_Mut3.bw |
196.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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