GSM7575320: Mouse.cortex_NeuN.pos; Mus musculus; RNA-Seq
Nuclei isolation from mouse cortical tissues was performed as described with minor modifications (Hu* & Fabyanic* et al., 2017; PMID: 29220646). Briefly, mouse brains were rapidly resected on ice. Cortices were flash-frozen in liquid nitrogen and stored at -80°C before nuclear isolation. Next, 14mL sucrose cushion (1.8M sucrose (Sigma-Aldrich, RNase and DNase free, ultra-pure grade), 10mM Tris-HCl pH 8.0, 3mM MgAc2 (Sigma-Aldrich), and protease inhibitor cocktail (Roche)) was added to the bottom of centrifuge tubes (Beckman Coulter). Using a glass homogenizer (Wheaton), a frozen mouse cortical sample was dounce homogenized (19 times with loose pestle followed by 4 times with tight pestle) in 12mL homogenization buffer (0.32M sucrose, 5mM CaCl2 (Sigma-Aldrich), 3mM MgAc2, 10mM Tris-HCl pH 8.0, 0.1% Triton X-100 (Sigma-Aldrich), 0.1mM EDTA (Invitrogen), protease inhibitor cocktail (Roche)). Homogenates (~12ml) were carefully layered onto the sucrose cushion in the centrifuge tubes, and 10mL homogenization buffer was added atop of the homogenates. The tubes were then balanced and centrifuged in a Beckman Coulter L7-65 Ultracentrifuge at 82,705 g at 4°C for 2hr using a Beckman Coulter SW28 swing bucket rotor (Beckman Coulter). The supernatant was carefully removed via aspiration. 1ml chilled DPBS was added to resuspend the nuclear pellet, and nuclei were subsequently transferred to a 1.5-mL tube. Nuclei were pelleted at 500 g for 10 min at 4C, 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 FANS. Drop-seq platform based single-nucleus RNA-sequencing (snRNA-seq/sNucDrop-seq) was performed as previously reported with some modifications related to NeuN antibody based nuclei isolation (Hu* & Fabyanic* et al., 2017; PMID: 29220646). Briefly, FANS-sorted NeuN-positive and NeuN-negative nuclei were individually diluted to a concentration of 100 nuclei/mL in DPBS containing 0.01% BSA. Approximately 1.25 mL of this single-nucleus suspension was loaded for each sNucDrop-seq run. The single-nucleus suspension was then co-encapsulated with barcoded beads (ChemGenes) using an Aquapel-coated PDMS microfluidic device (mFluidix) connected to syringe pumps (KD Scientific) via polyethylene tubing with an inner diameter of 0.38mm (Scientific Commodities). Barcoded beads were resuspended in lysis buffer (200 mM Tris-HCl pH8.0, 20 mM EDTA, 6% Ficoll PM-400 (GE Healthcare/Fisher Scientific), 0.2% Sarkosyl (Sigma-Aldrich), and 50 mM DTT (Fermentas; freshly made on the day of run) at a concentration of 120 beads/mL. The flow rates for nuclei and beads were set to 4,000 mL/hr, while QX200 droplet generation oil (Bio-rad) was run at 15,000 mL/hr. A typical run lasts 20 min. Droplet breakage with Perfluoro-1-octanol (Sigma-Aldrich), reverse transcription and exonuclease I treatment were performed, as previously described, with minor modifications7. For up to 120,000 beads, 200 μL of reverse transcription (RT) mix (1x Maxima RT buffer (ThermoFisher), 4% Ficoll PM-400, 1 mM dNTPs (Clontech), 1 U/mL RNase inhibitor, 2.5 mM Template Switch Oligo (TSO: AAGCAGTGGTATCAACGCAGAGTGAATrGrGrG) (Macosko et al., 2015), and 10 U/ mL Maxima H Minus Reverse Transcriptase (ThermoFisher)) were added. The RT reaction was incubated at room temperature for 30min, followed by incubation at 42C for 120 min. To determine an optimal number of PCR cycles for amplification of cDNA, an aliquot of 6,000 beads was amplified by PCR in a volume of 50 μL (25 μL of 2x KAPA HiFi hotstart readymix (KAPA biosystems), 0.4 μL of 100 mM TSO-PCR primer (AAGCAGTGGTATCAACGCAGAGT (Macosko et al., 2015), 24.6 μL of nuclease-free water) with the following thermal cycling parameter (95C for 3 min; 4 cycles of 98C for 20 sec, 65C for 45 sec, 72C for 3 min; 9 cycles of 98C for 20 sec, 67C for 45 sec, 72C for 3 min; 72C for 5 min, hold at 4C). After two rounds of purification with 0.6x SPRISelect beads (Beckman Coulter), amplified cDNA was eluted with 10 μL of water. 10% of amplified cDNA was used to perform real-time PCR analysis (1 μL of purified cDNA, 0.2 μL of 25 mM TSO-PCR primer, 5 μL of 2x KAPA FAST qPCR readymix, and 3.8 μL of water) to determine the additional number of PCR cycles needed for optimal cDNA amplification (Applied Biosystems QuantStudio 7 Flex). We then prepared PCR reactions per total number of barcoded beads collected for each sNucDrop-seq run, using 6,000 beads per 50-μL PCR reaction, and ran the aforementioned program to amplify the cDNA for 4 + 10 to 12 cycles. We then tagmented cDNA using the Nextera XT DNA sample preparation kit (Illumina, FC-131-1096), starting with 550 pg of cDNA pooled in equal amounts, from all PCR reactions for a given run. Following cDNA tagmentation, we further amplified the tagmented cDNA libraries with 12 enrichment PCR cycles using the Illumina Nextera XT i7 primers along with the P5-TSO hybrid primer (AATGATACGGCGACCACCGAGATCTACACGCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGT*A*C) (Macosko et al., 2015). After quality control analysis by Qubit 3.0 (Invitrogen) and a Bioanalyzer (Agilent), libraries were sequenced on an Illumina NextSeq 500 instrument using the 75-cycle High Output v2 Kit (Illumina). We loaded the library at 2.0 pM and provided Custom Read1 Primer (GCCTGTCCGCGGAAGCAGTGGTATCAACGCAGAGTAC) at 0.3 mM in position 7 of the reagent cartridge. The sequencing configuration was 20 bp (Read1), 8 bp (Index1), and 60 bp (Read2).