GSM8286261: OB, snRNA-Seq, replicate 1; Mus musculus; RNA-Seq
Tissue homogenization was carried out in a 7 ml Dounce tissue grinder. Frozen olfactory bulbs were transferred into 1 ml of ice-cold homogenization buffer (NIM 2), IGEPAL 0.4% (v/v), RNasIn 0.4 µl-1, SuperAsin 0.2 µl-1, 0.15 mM spermine, 0.5 mM spermidine). Homogenization was carried out by 10 strokes of loose pestle 'A' followed by 20 strokes of tight pestle 'B' after a 5 min incubation on ice. Homogenates were filtered through 20 µm cell strainers and centrifuged at 1000 g for 10 min at 4°C. Supernatant was removed by decanting and the nuclei pellets were resuspended in 1 ml FACS buffer (0.15 mM spermine, 0.5 mM spermidine, 0.2 U µl-1 protector RNase inhibitor, IGEPAL 0,4% (v/v), BSA 1% (wt/vol) and DAPI 1µM in RNAase-free PBS). 300 µl of suspension from each of three individual nuclear isolations were combined for one replicate and subjected to FACS. Cell-sorting was performed with FACSAria III system (BD Biosciences) using a 70 µm nozzle. Gating for forward and side scatter was used for doublet discrimination and sorting was performed selecting singlets in the DAPI+ population. 100.000 events were directly sorted into 10 µl of diluted nuclei buffer reaching a final concentration of ~1% BSA (wt/vol) and 0.2 U µl-1 protector RNase inhibitor. Libraries were prepared using the Chromium Next GEM Automated Single Cell 3' Reagent Kit v3.1 (10x Genomics, #1000268) following the manufacturer's instructions. Libraries were sequenced on a HiSeq4000 (Illumina) with 150-bp paired-end sequencing of read 2