Illumina HiSeq 2000 sequencing; GSM2079127: 1772099-104_A01; Mus musculus; RNA-Seq

1772099-104_A01

We explore the heterogeneity of mouse thoracic ganglia demonstrating the presence of an unexpected variety of cell-types and identify specialized populations of nipple- and pilo-erector muscle neurons. These neurons extend axonal projections and are born amongst other neurons during embryogenesis, but remain unspecialized until target organogenesis occurs postnatally. Target innervation and cell-type specification is coordinated by an intricate acquisition of unique combinations of growth factor receptors and the initiation of expression of concomitant ligands by the nascent erector muscles. Overall design: RNA-seq analysis of 298 single sympathetic neuronal cells from the mouse thoracic ganglion

Single cell transcriptome analysis of mouse thoracic sympathetic ganglia

Submitted by Gene Expression Omnibus on 02-SEP-2016

Sympathetic ganglia (SG) were collected in freshly oxygenated artificial cerebral spinal fluid (ASCF) kept on ice. SG were transferred into a 3cm plastic  dish  containing  2.7mL of 37°C digestion solution (500μL TrypLE™  Express (Life  Technologies), 2000μL Papain solution (Worthington Biochemical, 25U/ml), 100 μL DNaseI (Worthington  Biochemical, 1  mM) and 100 μL Collagenase/Dispase (Roche) 20 mg/ml). SGs were mechanically triturated  every 30 min  using glass  pasteur pipettes. After 1h incubation at 37°C, 100 μL fresh Collagenase/Dispase (20mg/mL) solution was added and SGs  were ripped open by using fine forceps. The solution was filtered using a 40μm cell strainer (FALCON) and collected in a 15 ml plastic tube. The digestion solution was diluted in 3ml ACSF and centrifuged at 100xg for 4 min at 4°C. The supernatant was removed and the pellet resuspended in 0.5 mL ASCF and 0.5 mL Neurobasal-A medium (Gibco). The cell suspension was transferred onto 80μl of Optiprep Density Solution (Sigma), 460 μL of ACSF and 460 μL Neurobasal media with supplements, and centrifuged at 100xg for 10min at 4°C and the solution concentrated to 100 μL by removing the supernatant. 10μL DNaseI was added to avoid cell clustering.  Cell density wasa djusted to 400-500 cells/μL. The Fluidigm C1 Autoprep System microfluidic chip (medium size) was used to capture cells (Zeisel et al., Science (2015) 6;347(6226):1138-42). Cells at the capture sites were visually inspected and only single healthy cells were selected for library preparation. Cell barcoding and fragmentation were performed in a single step using Tn5 DNA transposase as described previously (ibid.) but using a different Binding and Blocking buffer (10mM Tris, 250mM NalCl, 5mM EDTA, 0.5% SDS).

Single cell transcriptome analysis of mouse thoracic sympathetic ganglia

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