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| Status |
Public on Jun 18, 2024 |
| Title |
ChIP_AML_Rad21_28420_CTCF |
| Sample type |
SRA |
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| Source name |
hematopoietic
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| Organism |
Homo sapiens |
| Characteristics |
tissue: hematopoietic
cell type: AML blasts
genotype: RAD21 mut
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| 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.
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| 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.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixated using 0.5M DSG and 1% formaldehyde. Chromatin was sheared using Branson Sonifier 250. ChIP was performed with lysate of 2 Mio cells using a target specific antibody (anti-RAD21ab992 (abcam), anti-H3K27ac ab4729 (abcam), anti-CTCF 2899S (Cell Signaling), anti-STAG2 (custom produced), anti-STAG1 (custom produced), anti-MED12 (custom produced), anti-PU.1 sc352 (santa cruz)) and ProteinA Dynbeads. After de-crosslinking and proteinase K digest, DNA was purified using Monarch PCR & DNA clean-up kit (NEB).
Libraries were generated using the NEBNextUltra II DNA Library Prep Kit for Illumina with all available input material and 11 PCR cycles for amplification.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 550 |
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| Description |
19733
Allpat_CTCF.peaks.ann.filt.txt
RAD21mutvsCTRL.CTCF.Peaks_DESEQ.model.all.txt
EGAR00003766121
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| Data processing |
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
HOMER style tagDirectories were generated using HOMER makeTagDirectory (-checkGC and -unique). For AML samples, tag directories were corrected for copy number varations based on FreeC output genereated from DNA-Input-Sequencing data.
Bigwig coverage tracks were generated using HOMER’s makeUCSCfile function. For HSPC RAD21/CTCF/MED12/PU1/H3K27ac data sets, BigWigs were aditionally scaled based on median coverage across the top 5% common peaks detected in all samples.
For focal peaks, HOMER was used to call peaks (factor mode; -fdr 0.00001 -tbp). For region peaks, i.e. H3K27ac, HOMER was used for peak calling in region mode (-fdr 0.00001, -size 250, -L 0 -F 5 -minDist 350, -tbp -ntagThreshold 10). In case of AMLs, peak calling was performed in individual tag directories, in case of HSPCs condition-merged tag directories were used. For each data set, peak files of all conditions/groups were merged and filtered for low-mappability and blacklisted regions to generate reference peak sets. Subsequently, the individual samples were annotated to combined peak set to obtain peak coverage tables for each data set separetely.
Annotation tables with peak coverage were used for differential analysis using DESeq2 or edgeR to calculate differential Peak Coverage between controls and associated cohesin deficient samples. For H3K27ac data sets, counts were additionally normalized for length and GC content using offsets generated by the cqn R package, which were implemented in edgeR. For STAG1 and STAG2 HPSC datasets differential analysis was performed using HOMER’s getDiffExpression.pl routine with the -batch, -rlog and -normtotal options enabled. The same applies to RAD21 ChIP-seq in RAD21 KD HSPCs.
Assembly: GRCh38.p10
Supplementary files format and content: bigWig coverage track files for individual samples
Supplementary files format and content: tab-delimited file containing peak coordinates and coverage counts for each sample (HOMER annotation output)
Supplementary files format and content: tab-delimited file containing peak coordinates and DESeq2 or edgeR statistics for differential peak testing
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| Submission date |
May 21, 2024 |
| Last update date |
Jun 18, 2024 |
| Contact name |
Alexander Fischer |
| Organization name |
Leibniz Institute for Immunotherapy
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| Street address |
Franz-Josef-Strauß-Allee 11
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| City |
Regensburg |
| ZIP/Postal code |
93053 |
| Country |
Germany |
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| Platform ID |
GPL21697 |
| Series (2) |
| GSE268031 |
STAG2 Mutations Reshape the Cohesin-Structured Spatial Chromatin Architecture to Drive Gene Regulation in Acute myeloid Leukemia [ChIP-Seq] |
| GSE268035 |
STAG2 Mutations Reshape the Cohesin-Structured Spatial Chromatin Architecture to Drive Gene Regulation in Acute myeloid Leukemia |
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| Supplementary file |
Size |
Download |
File type/resource |
| GSM8284312_ChIP_AML_Rad21_28420_CTCF_CNVnorm.bigWig |
169.6 Mb |
(ftp)(http) |
BIGWIG |
| Raw data not provided for this record |
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