|
| Status |
Public on May 24, 2024 |
| Title |
shASH2L-A-H3K4me3 |
| Sample type |
SRA |
| |
|
| Source name |
SUM-44PE breast cancer cells
|
| Organism |
Homo sapiens |
| Characteristics |
passage: 70-85
source: pleural effusion cells from metastatic ER+ breast cancer patient
antibody: H3K4me3 (Abcam ab8580 lots GR273043-2, GR273043-3, GR75522-3
transduction: shASH2L
|
| Treatment protocol |
Target cells were transduced with virus in growth media supplemented with 5 μg/ml polybrene using 0.5-1.0 ml virus per 1 million cells. Cells were selected for resistance marker expression and maintained in puromycin-containing growth media beginning 24-48 hours after infection. Cells were cultured in selection media at least 5 days to allow expression of the construct. 3 μg/ml puromycin was used for selection and maintenance of shRNA-transduced SUM-44 cells.
|
| Growth protocol |
Cells were cultured at 37°C in 10% CO2 and passaged using Trypsin (Invitrogen Catalog # 15400-054) diluted to 1x with Hanks Buffered Salt Solution (Corning Cellgro Catalog #20-021-CV) supplemented with 1 ml/L sodium bicarbonate and 25 μg/ml gentamicin. SUM-44 cells were cultured in serum-free Ham’s F-12 medium supplemented with 1 μg/ml hydrocortisone, 1 mg/ml bovine serum albumin, 10 mM HEPES, 5 mM ethanolamine, 5 μg/ml transferrin, 10 nM triiodothyronine, 50 nM sodium selenite, 25 μg/ml gentamicin, 2.5 μg/ml fungizone, and 5 μg/ml insulin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
Sequencing libraries were prepared from ChIP DNA by the MUSC Genomics Core Facility with Rubicon ThruPLEX library preparation kits and sequenced as 35 bp single-end reads on an Illumina HiSeq 2500. Libraries for three biological replicates were prepared and sequenced for each sample type.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Illumina Casava1.8 software used for basecalling.
Sequenced reads (fastq files) were trimmed for adaptor sequence (Cutadapt), and masked for low-complexity or low-quality sequence (FASTQC)
Secondary analysis was carried out on an OnRamp Bioinformatics Genomics Research Platform (OnRamp Bioinformatics, San Diego, CA). OnRamp’s advanced Genomics Analysis Engine utilized an automated ChIPseq workflow to process the data, including data validation, quality control and read alignment to the HUMAN genome (hg19) using bowtie2
The resulting SAM files were sorted and inputted into the MACS2 peak calling program in the broad mode to generate peaks data including significance scoring.
MACS2 Peaks were mapped to genes using the HOMER Tools AnnotatePeaks.pl script.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited .txt files include MACS2 output for each Sample.
|
| |
|
| Submission date |
Jun 05, 2017 |
| Last update date |
May 24, 2024 |
| Contact name |
Gary Hardiman |
| E-mail(s) |
hardiman@musc.edu, g.hardiman@qub.ac.uk, glen@musc.edu
|
| Organization name |
Medical University of South Carolina
|
| Department |
Medicine
|
| Street address |
135 Cannon Street
|
| City |
Charleston |
| State/province |
SC |
| ZIP/Postal code |
SC |
| Country |
USA |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE99669 |
The 8p11-p12 amplicon oncogene ASH2L regulates expression of genes involved in tumorigenic processes via promoter H3K4me3 in luminal breast cancer. (SUM-44 ASH2L knockdown H3K4me3 ChIP-seq) |
|
| Relations |
| BioSample |
SAMN07193450 |
| SRA |
SRX2882769 |
