|
| Status |
Public on Jun 18, 2024 |
| Title |
ATAC_CD34_20_6_siCtrl |
| Sample type |
SRA |
| |
|
| Source name |
cord blood
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: cord blood
cell line: CD34_20
cell type: HSPC
genotype: wt
treatment: siCtrl
|
| Treatment protocol |
To obtain AML cells, PBMCS were extracted from bone marrow or whole blood samples of patients using Lymphoprep at the timepoint of first diagnosis and viably frozen. For knockdown experiments with healthy cells, HSPCs were transfected using the Neon electroporation system (Invitrogen, 1400V, 20ms, 2 pulses) at a density of 60,000 viable cells/µL with 1.33µg of chemically modified siRNAs (Axiolabs) per mio cells. Cells were kept in antibiotics-free media after transfection and harvested after 4d.
|
| Growth protocol |
CD34+ HSPCs were enriched from fresh cord blood samples on the day of birth using the CD34 MicroBead kit (Miltenyi biotec). Cells were expanded for 7d in serum-free Stem Span media (Stem Cell Technologies) supplemented with human recombinant cytokines (Peprotech): SCF (0.1ng/mL), FLT3 (0.1ng/mL), IL6 (0.004ng/mL), TPO (0.025ng/mL) and StemRegenin1 (7500nmol/mL, Stem Cell technologies) as well as Penicillin/Streptomycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
50k Cells were lysed using 0.1% NP-40 and 0.01% Digitonin. Tagment DNA TDE1 Enzyme (Illumina) was used to perform the tagmentation. DNA was purified using Monarch PCR & DNA clean-up columns (NEB)
Tagmented DNA was amplified using Phusion polymerase (NEB) and Nextera indexing primers (Illumina) by 11 cycles of PCR.
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| |
|
| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
| |
|
| Description |
CD34_combined.intersect.peaks.CD34.ann.table.txt
raw data cannot be deposited online
|
| Data processing |
Reads were trimmed for NEXTERA adapter sequences using Skewer (v0.2.2)
Sequences wer aligned to the human reference genome (GRCh38.p10) using bowtie2 (v. 2.4.5) in very sensitive mode
Samtools (v.1.6) was used to remove lower quality alignments with mapQ=<10 and sorting. Read positions in sam files were shifted to move the ends proximal to transposase cut sites (for positive strand: start +4 bp and its partner read start −5 bp, for negative strand: start −5 bp and its partner read start +4 bp)
HOMER style tagDirectories were generated using HOMER makeTagDirectory (-checkGC and -unique)
ATAC peaks were called in condition-merged tag directories using HOMER in region mode “-size 150 -minDist 250 -L 2 -fdr 0.00001" and factor mode "-size 250 -minDist 250 -L 2 -fdr 0.00001“. Only those region peaks, overlapping a focal peak using bedtools intersect were kept and used for the downstream analyses. HSPC peak sets were merged to create a common peak set. Individual HSPC tagdirectories were annotated to this combined peak set. AML tagdirectores were instead annotated to the combined AML RAD21 peak set to obtain cohesin-associated ATAC coverage tables.
Bigwig coverage tracks were generated using HOMER’s makeUCSCfile function. BigWigs of HSPCs were additionally scaled based on median coverage across the top 5% common ATAC peaks detected in all samples
Assembly: GRCh38.p10
Supplementary files format and content: bigWig coverage track files for individual samples
Supplementary files format and content: tab-delimited file containing ATAC or RAD21 peak coordinates and associated ATAC coverage counts for each sample (HOMER annotation output)
Supplementary files format and content: tab-delimited file containing qstat differential peak coverage results between control and either of the cohesin mutant groups (qstat)
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| |
|
| Submission date |
May 16, 2024 |
| Last update date |
Jun 18, 2024 |
| Contact name |
Alexander Fischer |
| Organization name |
Leibniz Institute for Immunotherapy
|
| Street address |
Franz-Josef-Strauß-Allee 11
|
| City |
Regensburg |
| ZIP/Postal code |
93053 |
| Country |
Germany |
| |
|
| Platform ID |
GPL21697 |
| Series (2) |
| GSE267678 |
STAG2 Mutations Reshape the Cohesin-Structured Spatial Chromatin Architecture to Drive Gene Regulation in Acute myeloid Leukemia [ATAC-Seq] |
| GSE268035 |
STAG2 Mutations Reshape the Cohesin-Structured Spatial Chromatin Architecture to Drive Gene Regulation in Acute myeloid Leukemia |
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