title: GSM8337801: Interneurons, Saline, PND 15, 2 biol rep pooled; Mus musculus; RNA-Seq
library_construction_protocol: Sensory cortex was dissected from the ipsilateral hemisphere. Tissue was snap frozen on dry ice and stored at -80° Celsius for 1 week. One hemicortex sample from a male and female mouse of the same condition were pooled together for simultaneous nuclei dissociation. RNAse-free instruments and surfaces, tissue was mechanically dissociated using a 2 mL glass tissue homogenizer (Kontes Glassware) on ice using 5 strokes of the loose pestle (A) and 7 strokes of the tight pestle (B) in nuclei isolation media (250 mM sucrose, 25 mM KCl, 5 mM MgCl2, 10 mM Tris pH 8.0, 0.2 U/uL of Protector RNase inhibitor (Roche), and nuclease free H2O). Dissociated nuclei were pelleted in an Eppendorf tube (1000 g, 5 minutes, 4°C) and gently resuspended in nuclei storage buffer (166.5 mM sucrose, 5 mM MgCl2, 10 mM Tris pH 8.0, 0.2 U/uL of Protector RNase inhibitor and nuclease free H2O) using a P1000 pipette. Nuclei were again pelleted (1000 g, 5 minutes, 4°C) and resuspended in blocking buffer (1% (weight/volume) RNase-free BSA in nuclei storage buffer). Hoechst was added to samples immediately prior to sorting and no immunostaining was performed. Nuclei were sorted on a BD FACSAria Fusion, gated for Hoechst+, single, GFP+ nuclei. Each sorted sample contained 20,000 GFP+ nuclei sorted directly into a 1.5 ml BSA-coated, lubricated Eppendorf tube. No post-sort centrifugation was performed to avoid aggregation and preserve nuclei integrity. The final sort volume was loaded directly into the 10X Genomics Chip. 20,000 sorted nuclei per sample were loaded per well of the Chromium Next GEM Chip G (10X Genomics) and libraries were generated using the Next GEM Single Cell 3' reagent kit v3.1 (10X Genomics) according to the manufacturer's instructions. snRNA-seq