title: GSM8831219: Dorsal horn neurons, ipsilateral, heat #1; Mus musculus; RNA-Seq
library_construction_protocol: Mice with a Cre dependent red fluorescent protein variant tdTomato reporter (10-20 weeks old, both males and females) were sacrificed with over dosage of isoflurane. Lumbar spinal cord was dissected out immediately and kept in freshly oxygenated artificial cerebrospinal fluid solution (modified ACSF: 87 mM NaCl, 2.5 mM KCl, 1.25 mM NaH2PO4, 26 mM NaHCO3, 75 mM Sucrose, 10 mM Glucose, 0.5 mM CaCl2, 4 mM MgSO4) on ice. Gray matter from lumbar spinal dorsal horn (or only lumbar 4-6 from ipsilateral side) was dissected out and transferred to a plastic petri dish (3 cm) with 2 mL pre-warmed digestion enzyme in ACSF solution (Papain, 25 unit/mL and DNase I, 55 unit/mL, Worthington Biochemical). For spinal dorsal horn neuronal atlas construction, each experiment included 2-3 mice (BAF53b-Cre*R26Tom). For labeled active ensembles (FosdsTVA*R26Tom) or neurons from Sni mice (BAF53b-Cre*R26Tom), 4 mice were included for each suspension experiment. The following single cell suspension was performed according to our previous protocol (6) with modifications. Briefly, spinal cord tissues were cut into pieces and triturated (10 x up and down) every 10 min (4 x) using glass Pasteur pipettes with decreasing diameter (pre-coated with 0.5% BSA). Cell suspension was filtered through a 30 µm cell strainer (CellTrics, Sysmex) and washed with additional 1.5 mL ACSF and 0.5 mL PBS. Cells were spin down with centrifugation (300 g x 5 min, 4 °C) and resuspended with 2 mL ACSF. Cell suspension was carefully loaded on top of the same volume of OptiPrep density gradient medium (5.4% iodixanol in ACSF, Sigma) and centrifuge with 300 g x 10 min at 4 °C. Gradient centrifugation step was replaced with centrifugation (300 g x 5 min, 4 °C) for some samples. Cell pellet was resuspended with 4 mL ACSF. SYTOX Blue (Invitrogen, ThermoFisher Scientific) was added to stain dead cells. Then tdTomato positive and SYTOX Blue negative cells were sorted with fluorescence activated cell sorting (FACS) cell sorters (BD FACSAria Fusion / BD FACSAria III) at 4 °C. Cells were concentrated when necessary by centrifugation (300 g x 5 min, 4 °C) and resuspended with proper volume (~1000 cells/ µl) of ACSF solution. Sorted cells were loaded onto the 10X Chromium chip G to yield single cell droplet with v3 or v3.1 kit (10X genomics). Targeted cell recovery for neuronal atlas construction or Sni samples was settled to 5000 cells, whereas the samples from captured active ensembles were aiming for 1000 cells. Reverse transcription, cDNA amplification and library construction were performed according to the user guide provided by manufacture.