title: GSM1263876: proximal cell 7; Mus musculus; RNA-Seq
library_construction_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)
attributes.tag: GEO Accession
attributes.value: GSM1263876