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Status |
Public on Sep 30, 2021 |
Title |
Ex_vivo_Meg_day_4 scRNA-seq |
Sample type |
SRA |
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Source name |
CD34_RO03183
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Organism |
Homo sapiens |
Characteristics |
cell type: CD34+ differentiation day: 4
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Growth protocol |
We used a 2-step erythroid differentiation protocol as described in Giarratana et al., 2011, Blood. Briefly, frozen CD34+ HSPCs are conditioned overnight in serum-free media in the presence of FLT3, TPO and IL3. Cells were then transferred in Step 1, serum-free media supplemented with BSA, Insulin, human-holotransferrin, heparin, hydrocortisone, human recombinant IL-3, hrEPO, and hrSCF and maintained for 7 days. On day 8, cells were transferred to Step 2 media where we withdraw hydrocortisone and IL-3 and maintain the rest of the components. Cells are maintained in Step 2 media until culture termination on day 12. CD34+ HSPCs were differentiated to the megakaryocyte lineage by culturing for 11 days in IMDM based media containing 30ng/mL rhTPO , 1ng/mL rhSCF , 7.5ng/mL rhIL-6 , 13.5ng/mL rhIL-9 , 20% BIT , 40µg/mL LDL , 0.05mM beta mercaptoethanol
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Extracted molecule |
total RNA |
Extraction protocol |
Frozen culture cell suspension from ex vivo erythroid or megakaryocytic culture or uncultured CD34+ were thawed and resuspended in PBS-BSA aiming for ~5,000 cell per sample Single cells were lysed in droplets using the 10X Chromium instrument Reverse transcription was performed in droplet and library construction was performed according to the 10X Chromium Single Cell 3' Reagent Kit User Guide Rev A
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
Illumina HiSeq 4000 |
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Description |
10X Single Cell 3' Reagent Kit V2
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Data processing |
Library strategy: scRNA-seq FASTQ files were demultiplexed using cellranger demux with default parameters BAM files were generated using 10X Genomics Cellranger V1.3 using default parameters Gene-barcode (count) matrices were generated using 10X Genomics Cellranger V1.3 using default parameters Genome_build: GRCh38 Supplementary_files_format_and_content: Cell barcodes in tab separated file Supplementary_files_format_and_content: Gene list in tab separated file Supplementary_files_format_and_content: Cell-gene count matrix .mtx formatted
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Submission date |
Sep 02, 2021 |
Last update date |
Sep 30, 2021 |
Contact name |
Grigorios Georgolopoulos |
E-mail(s) |
ggeorgol@altius.org
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Organization name |
Altius Institute
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Street address |
2211 Elliott Ave #400
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City |
Seattle |
State/province |
WA |
ZIP/Postal code |
98121 |
Country |
USA |
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Platform ID |
GPL20301 |
Series (2) |
GSE182816 |
Discrete regulatory modules instruct hematopoietic lineage commitment and differentiation |
GSE183267 |
Discrete regulatory modules instruct hematopoietic lineage commitment and differentiation [scRNA-seq] |
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Relations |
BioSample |
SAMN21206610 |
SRA |
SRX11996086 |
Supplementary file |
Size |
Download |
File type/resource |
GSM5554287_Ex_vivo_Meg_day_4.barcodes.tsv.gz |
21.1 Kb |
(ftp)(http) |
TSV |
GSM5554287_Ex_vivo_Meg_day_4.genes.tsv.gz |
484.6 Kb |
(ftp)(http) |
TSV |
GSM5554287_Ex_vivo_Meg_day_4.matrix.mtx.gz |
29.3 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
Raw data are available in SRA |
Processed data provided as supplementary file |
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