GSM2845368: nuclei-ctx-8; Mus musculus; RNA-Seq
Mouse brains (postnatal 6-10 weeks) were rapidly resected on ice. Cortices were freshly processed or flash frozen in liquid nitrogen for 2 minutes and subsequently kept at -80°C before nuclear isolation. Briefly, 14 mL of sucrose cushion was added to the bottom of centrifuge tubes (Beckman Coulter). Using a glass homogenizer (Wheaton), a freshly isolated or frozen mouse cortex sample was subjected to dounce homogenization (21 times with loose pestle followed by 7 times with tight pestle) in 12 mL of homogenization buffer. For in vitro cultured cells, cell pellets (~5 million cells) were resuspended in homogenization buffer and dounced 20 times with a loose pestle. Homogenates (~12 mL) were layered onto the sucrose cushion in the centrifuge tubes, and 10 mL of homogenization buffer was added atop of the homogenates. The tubes were then centrifuged in a Beckman Coulter L7-65 Ultracentrifuge at 25,000 rpm at 4°C for 2 hours using a Beckman Coulter SW28 swinging bucket rotor (Beckman Coulter). The supernatant was carefully removed via aspiration. 1 mL of chilled DPBS with protease and RNase inhibitor (Lucigen) was added to resuspend the nuclear pellet, and nuclei were subsequently transferred to a 1.5-mL tube. Nuclei were pelleted at 5,000 rpm for 10 min at 4°C, and then resuspended in 0.01% BSA (Sigma-Aldrich) in DPBS. After resuspension, nuclei were filtered through a 40-μm cell strainer (Fisher Scientific), visually inspected for morphology and quality assurance, and counted using a Fuchs-Rosenthal counting chamber before droplet microfluidic encapsulation. Single cell or nucleus RNA sequencing was performed using the Drop-Seq method. Briefly, single cells, polyA-containing barcoded beads (ChemGenes), and droplet generation oil (Bio-Rad) were each flowed into individual input ports on a microfluidic device (uFluidix). Cells in droplets were lysed and hybridized to beads. Beads were isolated and subjected to reverse transcription (Thermo Fisher) and exonuclease I (NEB) treatment, followed by PCR of full length transcripts (Kapa). Final libraries were created by tagmentation of cDNA (Illumina), and amplified by PCR and purified by SPRI beads.