GSM8008766: Control, replicate 2, snRNAseq; Mus musculus; RNA-Seq
30 mg dounce homogenized hippocampus tissue was resuspended in 500 µL of nuclei permeabilization buffer (0.1% Triton-X-100 (Sigma-Aldrich, T8787), 1X protease inhibitor, 1 mM DTT, and 0.2 U/µL RNase inhibitor (Promega, N211B), 2% BSA (Sigma-Aldrich, SRE0036) in PBS). Sample was incubated on a rotator for 5 min at 4°C and then centrifuged at 500 rcf for 5 min (4°C, run speed 3/3). Supernatant was removed and pellet was resuspended in 400 µL of sort buffer (1 mM EDTA 0.2 U/µL RNase inhibitor (Promega, N211B), 2% BSA (Sigma-Aldrich, SRE0036) in PBS) and stained with DRAQ7 (1:100; Cell Signaling, 7406). 75,000 nuclei were sorted using a SH800 sorter (Sony) into 50 µL of collection buffer consisting of 1 U/µL RNase inhibitor in 5% BSA; the FACS gating strategy sorted based on particle size and DRAQ7 fluorescence. Sorted nuclei were then centrifuged at 1000 rcf for 15 min (4°C, run speed 3/3) and supernatant was removed. Nuclei were resuspended in 35 µL of reaction buffer (0.2 U/µL RNase inhibitor (Promega, N211B), 2% BSA (Sigma-Aldrich, SRE0036) in PBS) and counted on a hemocytometer. 12,000 nuclei were loaded onto a Chromium Controller (10x Genomics) Libraries were generated using the Chromium Single-Cell 5' HT Library Construction Kit v2 with the Chromium Single-Cell B Chip Kit (10x Genomics, 1000153) and the Chromium i7 Multiplex Kit for sample indexing (10x Genomics, 120262) according to manufacturer specifications. CDNA was amplified for 12 PCR cycles. SPRISelect reagent (Beckman Coulter, B23319) was used for size selection and clean-up steps. Final library concentration was assessed by Qubit dsDNA HS Assay Kit (Thermo-Fischer Scientific) and fragment size was checked using Tapestation High Sensitivity D1000 (Agilent) to ensure that fragment sizes were distributed normally about 500 bp. Libraries were sequenced using the NextSeq500 and a NovaSeq6000 (Illumina) with these read lengths: 28 + 8 + 91 (Read1 + Index1 + Read2).