GSM9248579: Chronic-Adult-Sox11; Mus musculus; RNA-Seq
For extraction of nuclei, frozen tissue was promptly transferred from -80 °C storage to a chilled 2mL dounce (KIMBLE, Ref. DWK885300-0000) with chilled Nuclei EZ Lysis Buffer (Sigma-Aldrich N 3408). The sample was dounced 25 times with pestle A and 20 times with pestle B. The dounced sample was then transferred to a chilled 15mL conical tube and an additional 2mL of Nuclei EZ Lysis Buffer was added and gently mixed by inversion. The sample was incubated on ice for 5 minutes and centrifuged at 500G at 4 °C. The supernatant was removed, and the pellet was resuspended in 4mL of Nuclei EZ Lysis Buffer. The sample was incubated on ice and centrifuged at 500G at 4 °C for 5 minutes. Following the centrifugation, the supernatant was removed, and the pellet resuspended in 750 µl of Nuclei Suspension Buffer (NSB) (2% Bovine Serum Albumin (BSA, Sigma-Aldrich Ref. A9418), 40U/ µl RNase Inhibitor (Invitrogen Ref. AM2684), 1X PBS). For embryonic samples, which lacked AAV-derived nuclear mGl fluorescence propidium, iodide (Sigma, P4864, 1:500 dilution) was added to NSB to facilitate sorting. 5000 and 7000 nuclei were sorted directly into the Reverse Transcription Master Mix and single-nuclei libraries were prepared using Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (PN-1000269).