|
| Status |
Public on Jun 18, 2024 |
| Title |
HiC_AML_RAD21_26830 |
| Sample type |
SRA |
| |
|
| Source name |
hematopoietic
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: hematopoietic
cell type: AML blasts
genotype: RAD21 mut
|
| 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 |
2Mio Cells were fixated using 1% formaldehyde. Nuclei were extracted, a restriction digest using DpnII was performed, followed by incorporation of biotin-dATP and in situ proximity ligation using T4 DNA ligase. Ligated fragments were sheared on a Covaris S series sonicator and biotionylated fragments were purified using MyOne Streptavidin T1 Dynabeads.
Libraries were constructed on-bead following an in-house protocol with 12 cycles of PCR amplification using NEBNext High-Fidelity Q5 2x PCR Mix and Bioo Nextflex DNA sequencing adapters .
|
| |
|
| Library strategy |
Hi-C |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
12557
CohAML_CTRL_RAD21_merged.loop.scores.XYrm.txt
CohAML_CTRL_RAD21_merged.normTotalaverage.loop.scores.txt
RAD21mutvsCTRL.merged.loop.scores.DESeq.norm2Total.loopcoords2.txt
merged_rad21_mut_10kb.hic
EGAR00003764774
|
| Data processing |
Reads were trimmed for GATC sequences using Skewer (v0.2.2)
Sequences wer aligned to the human reference genome (GRCh38.p10) using bowtie2 (v. 2.4.5).
HOMER style tagDirectories were generated using HOMER makeTagDirectory (-checkGC and -unique) and filtered using “-restrictionSite GATC -both -genome hg38 -removePEbg -removeSelfLigation -removeSpikes 10000 5“ options
HOMERs findTADsAndLoops.pl was run to determine loop positions which were subsetquently merged to obtian consensus positions. Individual tag directories were scored for merged loop positions using findTADsAndLoops.pl -score function in default and normTotal mode. Sex chromosomes (X/Y) were filtered out after this step.
DESeq2 analysis was run on combined loop score tables of CTRL-AMLs vs. Either STAG2 or RAD21-mutant AMLs implementing a normalization factor calculated by dividing total scores from scoring to total counts by total scores derived from the regular interaction normalized scoring
For visualization of interactions, Hi-C data was analyzed using the FAN-C v.0.9.25 suite. The genome was binned at 10kb resolution and Hi-C matrices summarizing the number of valid pairs per bin were constructed and merged by patient group (.hic files)
Assembly: GRCh38.p10
Supplementary files format and content: Tab delimited file containing loop coordinates and loop scores for individual samples generated by HOMER with default HiC normalization (merged.loop.scores)
Supplementary files format and content: Tab delimited file containing loop coordinates and loop scores for individual samples generated by HOMER with normTotal HiC normalization (.normTotalaverage.merged.loop.scores)
Supplementary files format and content: Tab delimited file containing loop coordinates and DESeq2 differnetial loop statistics testing STAG2/RAD21-mutant AMLs vs CTRL-AMLs or cohesin depleted HSPCs (SA1/SA2/RAD21 KD) vs CTRL HSPCs (DESeq.norm2Total.loopcoords2.txt)
Supplementary files format and content: .hic file generated by FAN-C at 10 kb resolution
|
| |
|
| Submission date |
May 17, 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 |
GPL24676 |
| Series (2) |
| GSE267780 |
STAG2 Mutations Reshape the Cohesin-Structured Spatial Chromatin Architecture to Drive Gene Regulation in Acute myeloid Leukemia [Hi-C] |
| GSE268035 |
STAG2 Mutations Reshape the Cohesin-Structured Spatial Chromatin Architecture to Drive Gene Regulation in Acute myeloid Leukemia |
|
