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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Jul 01, 2020 |
| Title |
Mouse LSK [RPI2] |
| Sample type |
SRA |
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| Source name |
hematopoietic stem and progenitor cells with and without Bcor knockout
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| Organism |
Mus musculus |
| Characteristics |
cell type: hematopoietic stem and progenitor cells
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| Treatment protocol |
Briefly, bone marrow cells were extracted from C57BL/6 mice and cultured at a density of 1.5 x 10e6 in 96-round bottom plates with DC culture medium and 200 ng/ml Flt3L obtained from Dr Jian-Guo Zhang (WEHI) for experiments in Figure 1, and from BioXcell (#BE0098) for experiments in Figures 2-4. To generate Flt3-conditioned medium that can sustain the growth and differentiation of isolated DC progenitors, medium derived from Flt3L cultures after 3.5 days was centrifuged to remove cells, then passed through a 0.22-μm filter, and frozen aliquots stored at –80°C, and supplemented with additional Flt3L when thawed, as described previously.
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| Growth protocol |
All mice were bred and maintained under specific pathogen-free conditions at WEHI, according to institutional guidelines. C57BL/6 (CD45.2), C57BL/6 Pep3b (CD45.1), Cas9-Tg or Dach1-GFP mice aged between 8-16 weeks were used.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Bone marrow from C57BL/6-TG (UBC-GFP) was extracted and cells enriched for ckit (2B8 conjugated to APC) via magnetic activated cell sorting (MACS) positive selection using anti-APC beads (Miltenyi Biotec). The ckit-enriched fraction was subsequently stained for Sca-1 (E13 161-7 conjugated to Alexa Fluor 594 or Alexa Fluor 680), IL-7R (A7R34-2.2 conjugated to biotin with streptavidin-PECy7). C-kithi Sca1+IL-7R¬–CD11blo HSPCs were sorted using the single cell mode of a BD Influx, BD FacsARIA W or BD FacsAria L. Single cells, or 100-cell population controls, were sorted into 96 well round bottomed wells pre-seeded with 1.5 x 106 total C57BL/6 BM cells (filler cells) and cultured in Flt3L conditioned medium at 37 °C and 5% CO2 for 2.5 days.
For CEL-seq2, single cells were flow sorted into chilled 384-well PCR plates containing 1.2ul of primer/ERCC mix using a BD FACSAria III flow cytometer. Sorted plates were sealed and immediately frozen upside down at -80C. Then these plates, together with the mRNA mixture plates, were taken from -80C and processed using an adapted Cel-Seq2 protocol with the following variations. We pooled the samples after the first strand synthesis and the second strand synthesis was performed using NEBNext Second Strand Synthesis module. NucleoMag NGS Clean-up and Size select magnetic beads were used for all DNA purification and size selection steps.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
RPI2
cellular barcode: 8bp as in barcode_annotation.csv
umi position: 6bp after the cell barcode
barcode position: R1 first eight base
Index sorted hematopoietic stem and progenitor cells from Dach1 reporter mice, FACS value in Naik.SCORE_NN168_SeqPrimer layout_Nov19.xlsx
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| Data processing |
Gene counting matrix generated using scPipe 1.4.0.
Reads were aligned using Rsubread 1.29.6.
Genome_build: ENSEMBL Homo_sapiens.GRCh38.88
Supplementary_files_format_and_content: Gene counting matrix, rows represent genes and each column is a well.
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| Submission date |
Jul 01, 2020 |
| Last update date |
Jul 03, 2020 |
| Contact name |
Luyi Tian |
| E-mail(s) |
ltian@broadinstitute.org
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| Phone |
8577779729
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| Organization name |
Broad institute
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| Lab |
Fei Chen Lab
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| Street address |
415 main st Cambridge
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE153675 |
Clonal multi-omics reveals Bcor as a regulator of dendritic cell development and its action at a clonal level |
| GSE153676 |
Clonal multi-omics in DC development |
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| Relations |
| BioSample |
SAMN15420985 |
| SRA |
SRX8653260 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4649098_gene_count_RPI2.csv.gz |
1.1 Mb |
(ftp)(http) |
CSV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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