title: brt17_c4_cDNA
design: For sampling, we first transcranially perfused mice and sliced the brains in the ice-cold, oxygenized NMDG/HEPES solution. Coronal sections (300 μm) were prepared using a vibratome in ice-cold, oxygenized NMDG/HEPES solution. A macro-view zoom fluorescence microscope with CCD camera was used to check the expression of the helper virus and rabies virus. The starter regions were dissected out using a pair of titanium micro point under the microscope and transferred into digestion solution containing 2.5 mL oxygenized NMDG/HEPES solution, 10 units/ml papain and 1000 units/ml DNase I. The digestion was performed in temperature-controlled incubator at 37 ℃ using a shaker for a duration of 30 minutes. After digestion, we centrifuged the tissue at 400 g for 5 mins at 4 ℃ . After centrifugation, the digestion solution was replaced with 2 mL of resuspension solution containing oxygenized NMDG/HEPES solution, 2% FBS and 10 units/ml DNase I. In the resuspension solution, the tissue was gently broken down by repeatedly passing a glass straw with 300 μm diameter tip, followed by a glass straw with 200 μm diameter tip, until no tissue clumps were visible. To remove the potential tissue clumps, we used a pre-rinsed 40 μm cell strainer to filter the cell suspension and then refilled the filtered suspension to 10 mL with the resuspension. The cells were then washed by centrifuging at 400 g for 5 mins at 4 ℃ , and the resulting pellet was resuspended using 50 μL of a buffer consisting of oxygenated NMDG/HEPES solution and 2% FBS. The resulting cell suspension was prepared for DNB-C4 3.0 single-cell RNA sequencing.