 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 26, 2022 |
| Title |
SKNAS_T2_ATACseq |
| Sample type |
SRA |
| |
|
| Source name |
SK-N-AS
|
| Organism |
Homo sapiens |
| Characteristics |
lineage: Neuroblastoma
treatment: Tazemetostat
|
| Treatment protocol |
14 days treatment with tazemetostat or vehicle
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Briefly, 50,000 cells per replicate were pelleted and then resuspended and lysed in 50 uL ATAC resuspension buffer supplemented with 0.1% NP40, 0.1% Tween-20, and 0.01% Digitonin. After lysis, nuclei were pelleted and transposed using 2.5 uL Tn5 transposase in a 50 uL reaction.
Finally, the transposed DNA was purified using a commercial PCR cleanup kit and libraries were amplified and prepared for sequencing. Paired end sequencing (2x75) was performed on an Illumina NovaSeq.
|
| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
ATAC-Seq data analysis was performed in alignment with the ENCODE Consortium standards (https://www.encodeproject.org/atac-seq/). Briefly, the paired-end reads were trimmed and filtered using Trimmomatic v0.39 and mapped to the hg38 reference genome using bowtie2 with the –local –very sensitive –X 2000 and MAPQ > 5 options. Bam files were deduplicated using Picard. Only reads with MAPQ > 5 and mapping to chromosomes 1-22 and chrX were retained. Reads were shifted with 4 bp on the positive strand and -5 bp on the negative strand using AlignmentSieve available in deepTools v3.5.162. Replicate correlations were computed with the multiBamSummary and bamCorrelate tools available in deepTools and visualized as dendrograms and in PCA plots. Fragment size distributions were computed with the bamPEFragmentSize tool available in deepTools. Properly aligned reads for replicates were merged for each condition.
The merged reads for the four vehicle replicates were labeled “SKNAS_ATAC_CTRL” and similarly, the merged reads for the four tazemetostat treated replicates were labeled “SKNAS_ATAC_TAZE”. The mapped reads for individual and merged replicates were normalized in units of Reads Per Kilobase per Million (RPKM or rpm/bp) and coverage tracks for the RPKM signal were created as bigwig files for bins of size 20 base pairs by using the bamCoverage tool available in deepTools.
Peak calling was performed for properly aligned and for the merged reads with MACS2, with the –nomodel, --extsize to fragment length and –q 0.01 options. Area under curve (AUC) binding signal for peaks was computed with the bwtool64.
The binding sites identified by MACS263 on merged reads were aggregated into consensus peaks per condition by requiring AUC binding signal > 1000 for each replicate. The consensus peaks for both conditions were merged into a high confidence union peak set. A peak by sample counts matrix was created by counting the reads overlapping each peak with the multiBamSummary tool available from deepTools. The counts matrix was used to perform differential peak analysis in DESeq2. Differential binding across conditions was assessed based on the significance cut-offs |Delta(log2(AUC+1))| ≥ 0.5 and adjP-value ≤ 0.10. Genome track files were created with the bamCoverage tool available in deepTools and visualized as heatmaps created with the computeMatrix tool available in deepTools, and with the Integrative Genomic Viewer (IGV).
|
| |
|
| Submission date |
Jul 20, 2021 |
| Last update date |
Apr 29, 2022 |
| Contact name |
Nathaniel Wesley Mabe |
| E-mail(s) |
nathanielw_mabe@dfci.harvard.edu
|
| Organization name |
Dana-Farber Cancer Institute
|
| Department |
Pediatric Oncology
|
| Lab |
Kimberly Stegmaier
|
| Street address |
450 Brookline Avenue
|
| City |
Boston |
| State/province |
Massachusetts |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (2) |
| GSE180509 |
Epigenetic reversal of lineage plasticity enhances responsiveness to anti-GD2 therapy in neuroblastoma [ATAC-seq SK-N-AS] |
| GSE180516 |
Epigenetic reversal of lineage plasticity enhances responsiveness to anti-GD2 therapy in neuroblastoma |
|
| Relations |
| BioSample |
SAMN20331634 |
| SRA |
SRX11509341 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5464093_TAZE_2-coverage-tss-norm.bw |
184.5 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
|
|
|
|
 |
