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Sample GSM3739226 Query DataSets for GSM3739226
Status Public on Jun 01, 2019
Title amplicon.MF03.CALR
Sample type SRA
Source name MF03, amplicon, straight
Organism Homo sapiens
Characteristics source: PBMC
targeted mutation: CALR exon9 L367Rfs*46 (c.1099_1150del)
vaf (targeted exon sequencing): 20.6% (source: CD34- cells)
Sex: Male
age: 59
diagnosis: MF
wbc(k/mcl): 12.8
hg(g/dl): 12.7
platelets(k/mcl): 464
pb blast count: 0%
bm blast count: <5%
bm fibrosis: MF-3/3
treatment: Aspirin
Extracted molecule total RNA
Extraction protocol Cryopreserved patient samples approved by the Institutional Review Board of Memorial Sloan-Kettering Cancer Center 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. For the species mixing experiment, human MPL expressing Ba/F3 and UT-7 cell lines were generated by retroviral transduction, after which they were subjected to infection with CALR variant lentiviral supernatants. Wildtype UT7 cells and mutant Ba/F3 cells were mixed in equal proportions.
The standard 10x Genomics Chromium v.2 or v3 protocol was carried out according to manufacturer’s recommendations (10x Genomics, Pleasanton, CA) until after emulsion breakage and recovery of first strand cDNA. If the targeted gene of interest, e.g. SF3B1, was not robustly detected by the standard 10x procedure (i.e. if <60% of the expected cells showed expression) based on a priori knowledge in a similar dataset, a gene-specific 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 (Supplementary Table 3) 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 spiked into the remainder of the 10x library to be sequenced together on a HiSeq 2500 or sequenced separately on MiSeq with v3 chemistry (Illumina). The cycle settings were as follows: 26 cycles for read 1, 98 or 130 cycles for read 2, and 8 cycles for sample index.
Library strategy OTHER
Library source transcriptomic
Library selection other
Instrument model Illumina HiSeq 2500
Data processing 10x data was processed using Cell Ranger (version 2.1.0 or 3.0.0 according to used chemistry version) using default parameters. RNA reads were aligned to the human or mouse reference 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, and 5) Analyze reads with CB that are also observed in the 10x scRNA-seq data.
Output matrices processed from amplicon data have three columns: barcodes (BC), number of wildtype calls (, and number of mutant calls (
Supplementary_files_format_and_content: mutation call
Submission date Apr 29, 2019
Last update date Jun 02, 2019
Contact name Kyu-Tae Kim
Organization name New York Genome Center
Lab Landau Lab
Street address 101 Avenue of the Americas
City New York
State/province New York
ZIP/Postal code 10013
Country USA
Platform ID GPL16791
Series (2)
GSE117825 Amplicon of Single-Cell Genotyping of Transcriptomes
GSE117826 Single-Cell Genotyping of Transcriptomes
BioSample SAMN11533753
SRA SRX5767069

Supplementary file Size Download File type/resource
GSM3739226_MF03.CALR.txt.gz 40.8 Kb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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