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| Status |
Public on Jul 13, 2016 |
| Title |
distal cell 7 |
| Sample type |
SRA |
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| Source name |
HSPC-distal cell from col2.3GFP mouse
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| Organism |
Mus musculus |
| Characteristics |
cell type: distal
genotype/variation: col2.3GFP
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| Growth protocol |
Newborn col2.3GFP animals were injected with DiI-labeled LKS CD34-Flk2- adult bone marrow cells, as described below, and sacrificed 48 hours after transplantation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Femurs were dissected, embedded in 10% low melting temperature agarose (Lonza) and sectioned at 100 _ using a vibratome (Leica). Single OLC harvesting was performed using a physiology microscope BX51 (Olympus) equipped with filters to detect GFP and DiI fluorescence, DIC optics, micromanipulators (Eppendorff), real-time imaging camera, peristaltic pump, in-line heater, perfusion chamber (Harvard Apparatus) and SAS Air Syringe (Research Instruments). Sections were pre-screened for the presence of rare GFP-labeled OLCs located next to single DiI-positive transplanted HSCPs, which were found in 1-2 out of 15 sections per animal. Once a target proximal OLC was identified, the section was rotated so that the target was directly opposite the aspiration pipette (Humagen) and secured against the bottom of the perfusion chamber using a horizontal portion of the holding pipette (Humagen). With the aspiration pipette just above the target, the section was perfused with warm (37C) cell dissociation solution (Liberase TM, Roche) for 8-10 minutes while the target cell was visually monitored. Then, applying positive pressure from the micropipette using Air Syringe, hematopoietic cells surrounding the target OLC were dislodged to create a 20-30 _ clearing. Finally, the aspiration pipette was lowered onto the target OLC, the cell was gently detached from the endosteal surface and aspirated. The presence of GFP fluorescence in the aspirated cell inside the aspiration pipette was confirmed, the contents of the pipette was ejected into a PCR tube with the lysis buffer for the single cell RNA-Seq protocol, and frozen immediately at -80C.
Reverse transcription, cDNA amplification, library preparation and SOLiD RNA-Seq were performed as described in Tang, F. et al. mRNA-Seq whole-transcriptome analysis of a single cell. Nat Methods 6, 377-82 (2009)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
AB 5500 Genetic Analyzer |
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| Data processing |
reads were aligned against mm9 annotations (NCBI37.61) using tophat 1.2.0 with default settings
FPKM values were estimated using cufflinks 1.3.0 with default settings
Genome_build: mm9
Supplementary_files_format_and_content: tab-delimited text files, as written out by cufflinks
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| Submission date |
Nov 13, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Peter Kharchenko |
| Organization name |
Harvard Medical School
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| Department |
DBMI
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| Lab |
Kharchenko
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| Street address |
10 Shattuck St.
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL16790 |
| Series (1) |
| GSE52359 |
Single-cell measurements of osteolineage cells within the bone marrow cell niche |
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| Relations |
| BioSample |
SAMN02402482 |
| SRA |
SRX377502 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1263884_d7.genes.fpkm_tracking.gz |
775.3 Kb |
(ftp)(http) |
FPKM_TRACKING |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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