|
| Status |
Public on May 12, 2020 |
| Title |
EFKD_cnt_Rb_rep1 |
| Sample type |
SRA |
| |
|
| Source name |
A673 cells
|
| Organism |
Homo sapiens |
| Characteristics |
transduced shrna: iEF + empty cDNA
replicate: 1
|
| Treatment protocol |
Cells were retrovirally transduced with either control shRNA (iLuc) or shRNA targeting EWS/FLI (iEF) on day 0. Cells were selected in 2 ug/mL puromycin and transduced with either empty vector or cDNA encoding 3XFLAG-wtEWS/FLI on day 4. Cells were then double selected in puro and 100 ug/mL hygromycin B for 7 days before collection for CUT&Tag
|
| Growth protocol |
A673 cells were cultured in DMEM supplemented with FBS, PSQ, and NaPyruvate.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
NEBNext
For KD-rescue samples cells were seeded at 50% confluency and left to adhere overnight (day 0). shRNAs were retrovirally delivered on day 1. Selection in 2 µg/mL puromycin (Sigma P8833)until days 5, and then rescue or mock-rescue was performed. Cells were then double selected in puro and hygro for 7 days before collection for CUT&Tag.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
CUT&Tag
iEF_K27ac_cutoff_peaks.bed
iEF_LSD1_cutoff_peaks.bed
iEF_REST_cutoff_peaks.bed
|
| Data processing |
Library strategy: CUT&Tag
Illumina’s Bcl2fastq conversion software
FastQC
Quality and adapter trimming: TrimGalore!
Alignment to hg19 with bowtie2
SAM to BAM conversion with samtools
Peak calling with MACS2: first call peaks using both replicates of each condition pooled using "callpeak". Then use "bdgdiff" to call peaks up in test conditions as compared to Rb IgG. Peaks were included only if the summit in the spike-in normalized bigwig exceeded 10000.
BAM to BED and BED to spike-in normalized bedgraph with Pybedtools; spike-in normalization first normalized all samples to the same total number of reads and then to the number of spike-in reads.
Bedgraph to bigwig with bedGraphToBig UCSC util
Genome_build: hg19
Supplementary_files_format_and_content: bigwig, genome tracks for each sample
Supplementary_files_format_and_content: bed, peak calling output from MACS2 callpeak + bdgdiff
|
| |
|
| Submission date |
Feb 02, 2020 |
| Last update date |
Sep 30, 2021 |
| Contact name |
Emily Rose Theisen |
| E-mail(s) |
[email protected]
|
| Organization name |
Nationwide Children's Hospital
|
| Department |
Center For Childhood Cancer and Blood Diseases
|
| Street address |
700 Childrens Drive
|
| City |
Columbus |
| State/province |
OH |
| ZIP/Postal code |
43205 |
| Country |
USA |
| |
|
| Platform ID |
GPL20301 |
| Series (2) |
| GSE144651 |
Chromatin profiling of the relationship between EWS/FLI, LSD1, and H3K27 acetylation |
| GSE144688 |
Chromatin profiling reveals the relationship between EWS/FLI, LSD1, and the enhancer landscape in Ewing sarcoma |
|
| Relations |
| Reanalyzed by |
GSE185128 |
| BioSample |
SAMN13970071 |
| SRA |
SRX7661361 |
