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Status |
Public on Aug 07, 2023 |
Title |
M5 HSPCs, barcode-derived cDNA |
Sample type |
SRA |
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Source name |
Bone Marrow/Peripheral Blood
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Organism |
Homo sapiens |
Characteristics |
tissue: Bone Marrow/Peripheral Blood cell type: Hematopoietic Stem and Progenitor Cells (HSPCs)
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Extracted molecule |
genomic DNA |
Extraction protocol |
We performed DAbseq on unsorted mononuclear cells from 14 patients using a microfluidic approach with molecular barcode technology using the Tapestri platform (MissionBio) as previously described . Briefly, cryopreserved cells were blocked and stained with a pool of 22 oligo-conjugated antibodies. Individual samples were also incubated with unique anti-CD45 oligo-conjugated antibodies to provide sample-level identifiers, and groups of 2-3 individual patients were pooled together for multiplexed runs. Pooled samples were run through Mission Bio Tapestri platform as previously described. DNA from barcoded cells was amplified via PCR using a targeted panel that included 127 amplicons across 20 genes associated with acute leukemia. DNA PCR products were isolated and purified with standard library preparation protocol. Protein PCR products were isolated from the supernatant from AmpureXP bead purification via incubation with a 5’ Biotin Oligo (ITD), and purified using Streptavidin C1 beads.
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Library strategy |
OTHER |
Library source |
genomic |
Library selection |
other |
Instrument model |
Illumina NovaSeq 6000 |
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Description |
Mission Bio Tapestri
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Data processing |
FASTQ files from single-cell DAbseq samples were processed via an open-source pipeline. This analysis pipeline trims adaptor sequences, demultiplexes DNA panel amplicons and antibody tags into single cells, and aligns panel reads to the hg19 reference genome. Valid cell barcodes were called using the inflection point of the cell-rank plot in addition to the requirement that 60% of DNA intervals were covered by at least eight reads. Variants were called using GATK (v 4.1.3.0) according to GATK best practices. ITDseek was used to detect FLT3 internal tandem duplication (ITD) from amplicon sequencing reads. For each valid cell barcode, variants were filtered according to quality and sequence depth reported by GATK, with low quality variants and cells excluded based on the cutoffs of quality score < 30, read depth < 10, and alternate allele frequency < 20%. Cell-surface protein reads were normalized using centered log ratio transformation. To de-multiplex individual patients combined into a single sample, we used a novel computational approach incorporating both patient-specific anti-CD45 oligo-conjugated “hash” antibody tags as well as single nucleotide polymorphisms (SNPs) covered by the SC DNA panel. Individual samples were multiplexed together into groups of 3 and all SNPs were treated as binary (mutated or wildtype). For each multiplexed run, to identify SNPs that maximally differ between samples we filtered all SNPs mutated in <10% or >80% of cells. We performed a series of hierarchical clustering steps to further refine antibody hash data. Assembly: hg19 Supplementary files format and content: hdf5 files Supplementary files format and content: h5ad files Library strategy: DAbseq
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Submission date |
Apr 28, 2023 |
Last update date |
Aug 07, 2023 |
Contact name |
Catherine Smith |
E-mail(s) |
Catherine.Smith@ucsf.edu
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Organization name |
University of California, San Francisco
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Department |
Medicine
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Lab |
Smith Lab
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Street address |
513 Parnassus Avenue
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City |
San Francisco |
State/province |
California |
ZIP/Postal code |
94143 |
Country |
USA |
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Platform ID |
GPL24676 |
Series (2) |
GSE230827 |
Multiomic Single Cell Sequencing Identifies Stemlike Nature of Mixed Phenotype Acute Leukemia and Provides Novel Risk Stratification [DAb-seq] |
GSE232074 |
Multiomic Single Cell Sequencing Identifies Stemlike Nature of Mixed Phenotype Acute Leukemia and Provides Novel Risk Stratification |
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Relations |
BioSample |
SAMN34414801 |
SRA |
SRX20124080 |
Supplementary file |
Size |
Download |
File type/resource |
GSM7244783.hdf5 |
60.6 Mb |
(ftp)(http) |
HDF5 |
GSM7244783_M5_Ab.adata.h5ad.gz |
26.7 Mb |
(ftp)(http) |
H5AD |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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