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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 05, 2022 |
| Title |
CH03 10X |
| Sample type |
SRA |
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| Source name |
GCSF mobilized stem cells from patient
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| Organism |
Homo sapiens |
| Characteristics |
cell type: CD34+
tissue: Blood
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Cryopreserved patient samples approved by the Institutional Review Board of Dana Farber Cancer Institute were thawed and stained using standard procedures (10 min, 4°C) with the surface antibody CD34-PE-Vio770 (clone AC136, Miltenyi Biotec) and DAPI (Sigma-Aldrich). Cells were then sorted for DAPI-negative, CD34+ and CD34-negative cells using BD Influx.
The standard 10x Genomics Chromium v.3 protocol was carried out according to manufacturer’s recommendations (10x Genomics, Pleasanton, CA) until after emulsion breakage and recovery of first strand cDNA. A gene-specific DNMT3A primer was spiked into 10x primer mix at 1% of the concentration of the cDNA amplification primers for the initial cDNA PCR step. Then, during the amplification step, the 10x cDNA library underwent an extra cycle of PCR beyond the manufacturer’s recommended number of cycles. After cleanup with SPRIselect, a small portion of the cDNA library, 3 µL (~10% of total) was aliquoted for targeted genotyping, and the remaining cDNA underwent the standard 10x protocol. The cDNA set aside for GoT was amplified for 3 to 4 additional cycles using KAPA HiFi HotStart ReadyMix (KAPABiosystems) and 10x primer mix to provide sufficient material for the enrichment step. After clean-up, locus-specific reverse primers and the generic forward SI-PCR were used to amplify the site of interest of the cDNA template using ~10 PCR cycles. The locus-specific reverse primers contain a partial Illumina read 2 handle, a stagger to increase the complexity of the library for optimal sequencing and a gene specific region to allow specific priming. The SI-PCR oligo (10x) anneals to the partial Illumina read 1 sequence at the 3’ end of the molecule, preserving the cell barcode (CB) and UMI. After the initial amplification and SPRI purification to remove unincorporated primers, a second PCR was performed with a generic forward PCR primer (P5_generic) to retain the CB and UMI together with an RPI-x primer (Illumina) to complete the P7 end of the library and add a sample index. The targeted amplicon library was subsequently sequenced separately. The cycle settings were as follows: 28 cycles for read 1, 98 or 130 cycles for read 2, and 8 cycles for sample index.
Single-cell RNA-seq (10X) with targeted amplification of a mutation of interest.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
10x data was processed using Cell Ranger 3.0.1 using default parameters. RNA reads were aligned to the human or mouse referece genome, or human and mouse reference concatenation for species mixing experiment.
Amplicon reads was processed following steps: 1) Identification of reads with proper priming, 2) Identification of cell barcodes (CB) that match with the whitelisted CB, 3) Replacement of CB that are not perfectly matched with the whitelist CB, 4) Deduplication of reads, 5) Analyze reads with CB that are also observed in the 10x scRNA-seq data, 6) Collapse networks of closely related UMIs within CBs to correct for sequencing error, 7) Matching of CB/UMI pairs found in 10x scRNA-seq data, discarding those matching non-DNMT3A genes and those matching no gene below a supporting read threshold.
Genome_build: hg19
Supplementary_files_format_and_content: Processed 10X data is matrix table of gene counts per cell for each sample
Supplementary_files_format_and_content: Output matrices processed from amplicon data have three columns: barcodes (BC), number of wildtype calls (num.WT.call), and number of mutant calls (num.MUT.call).
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| Submission date |
Sep 16, 2020 |
| Last update date |
Jul 05, 2022 |
| Contact name |
Neville Dusaj |
| E-mail(s) |
ned2010@med.cornell.edu
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| Organization name |
Weill Cornell Medicine
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| Lab |
Landau Lab
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| Street address |
413 E 69th St.
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10021 |
| Country |
USA |
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| Platform ID |
GPL24676 |
| Series (1) |
| GSE158067 |
Single-cell multi-omics in human clonal hematopoiesis reveals that DNMT3A R882 mutations perturb early progenitor states through selective hypomethylation. |
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| Relations |
| BioSample |
SAMN16182251 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4790030_CH03.barcodes.tsv.gz |
56.9 Kb |
(ftp)(http) |
TSV |
| GSM4790030_CH03.features.tsv.gz |
302.7 Kb |
(ftp)(http) |
TSV |
| GSM4790030_CH03.matrix.mtx.gz |
89.5 Mb |
(ftp)(http) |
MTX |
| Processed data are available on Series record |
| Raw data not provided for this record |
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