title: GSM5509854: C3; Homo sapiens; RNA-Seq
library_construction_protocol: Single nuclei isolation from post-mortem frozen human brain tissue  Protocol based on (Krishnaswami et al 2016). All steps and performed on ice, in RNase free conditions.    Frozen brain tissue blocks were stored at -80°C. The tissue is first attemperated at -12°C to enable dissection. Small pieces between (60-200 mg) of the region of interest are dissected encompassing an equal representation of all cortical layers. The tissue sample is chopped into small fragments using a chilled scalpel and immediately transferred to a Dounce grinder (7mL, clearance 0.020 to 0.056 mm) with 2.4 mL of homogenization buffer (Tris pH8 10 nm, MgCl2 5 mM, KCl 25 mM, Sucrose 250 mM, DTT 1 μM, Protease Inhibitor 0.5x [cOmplete, Roche #4693159001], RNase inhibitor 0.2 U/μl [NxGen, Lucigen #30281], Triton-x100 0.1% v/v). While one ice, the tissue is homogeinized with 30 firm grinder strokes. The homogenate is filtered through a 40 μm Corning cell strainer into two 1.5 mL Eppendorf tubes and its quality assessed on a hemocytometer. After centrifuging the homogenate at 1000g for 8 minutes at 4°C, the supernatant is aspirated and the pellets gently resuspended in 200 μl of cold homogenization buffer. The homogenate is pooled and the total volume measured (should be ~450 μl). An exactly equal volume (~450 μl) of 50% v/v iodixanol is added and gently mixed with a pipette, for a final 25% iodixanol concentration. The mixture is transferred to a new 2 mL Eppendorf tube containing 900 μl of cold iodixanol 29%, slowly layering it over. Then, the tubes are centrifuged at 13500g for 20 min at 4°C. The supernatant is removed and discarded without disrupting the pellet, and avoiding contamination from the top layer. The pellet containing the nuclei is detached with a small amount of immunostaining buffer (PBS pH 7.4, BSA 0.05%, MgCl2 5 mM, DNAse I 2U/mL, RNase inhibitor 0.2 U/μl), transferred to a new tube and gently resuspended in a total volume of 500 μl of immunostaining buffer. After 15 min incubation, the primary antibody is added (Mouse anti NeuN, 1:1000; Millipore #MAB377) and the suspension incubated for 40 min at 4°C with rotation. After incubation an equal volume (500 μl) of immunostaining buffer is added and the tube inverted several times, followed by centrifugation at 500g for 5 min at 4°C. The supernatant is carefully removed and the pellet resuspended in 500 μl of immunostaining buffer. After adding the secondary antibody (goat anti mouse, Alexa Fluor 647, 1:1000) and nuclear staining (Hoescht 33258, 5 μg/mL), the tube is incubated for 30 min at 4°C with rotation. Before bringing the sample for sorting, its quality is checked on a hemocytometer, expecting a yield between 1 and 5 million nuclei per milliliter.    Fluorescence Activated Cell Sorting (FACS) of neuronal and non-neuronal nuclei    Immunostained nuclei suspension is sorted using FACS to obtain a clean single-nuclei suspension enriched for the population of interest.    Nuclei were sorted with a BD SORP FACSAria II at the Janis V. Giorgi Flow Cytometry Core Laboratory at UCLA. The settings and calibrations are as follows: PBS was used as the sheath fluid, with a sheath pressure of 20 psi. A 100 μm nozzle tip was used, and the frequency of droplet generation was ~30kHz. The primary laser was a blue Trigon 488-nm, used in the generation of forward scatter (FSC) and side scatter (SSC). The secondary lasers were a UV Trigon 355-nm and red Trigon 640-nm, used for the excitation of Hoechst 33258 and Alexa Fluor 647, respectively. Sample events were acquired at <30% of droplet occupancy. Sort decision gates were based on a combination of regions drawn around target populations in several 2D plots: FSC height versus SSC height: SSC area versus FSC width: FSC area versus FSC width: Hoechst fluorescence (bandpass filter 450/50) versus Alexa Fluor 647 fluorescence (bandpass filter 670/30). Hoechst fluorescence was used to discriminate singles from nuclei aggregates and debris. Alexa Fluor 647 was used to discriminate neuronal nuclei (NeuN positive) from non-neuronal nuclei (NeuN negative). Two populations were collected serially, the first corresponding to neuronal nuclei and the second one corresponding to neuronal nuclei plus non-neuronal nuclei. For each population, 3.2*105 nuclei are collected in a 1.5 mL Eppendorf tube containing 200 μl of collection buffer (PBS pH 7.4, RNase inhibitor 1 U/μl). After collection, BSA 10% is added to the tube for a final concentration of 0.01% and the tube inverted several times. Drop-Seq was performed largely as described previously (Macosko et al., 2015). Few modifications were made to the original protocol to accommodate single nuclei, which contain small amounts of RNA and are more resistant to surfactant-induced lysis. Reducing droplet size and increasing surfactant concentration in lysis buffer gave satisfactory results.    The sorted nuclei were kept in ice and processed for Drop-Seq between 1 h and 1.5 h after collection. Final nuclei concentration was 180 nuclei/μl, for a droplet occupancy of ~5%. Barcoded beads were diluted in lysis buffer containing 1% Sarkosyl (6% Ficoll PM-400, 1% Sarkosyl, 20mM EDTA, 0.2M Tris pH 7.5, 50 mM DTT) to a final concentration of 230 beads/μl, for a droplet occupancy of ~7%. Droplets were generated using the microfluidics device from Macosko et al., 2015 with flow rates of 3 mL/h for each aqueous suspension and 16 mL/h for the droplet generation oil (QX200 Oil for EvaGreen, Bio Rad). About 1 mL of each aqueous solution was flowed, generating droplets of 100-105 μm of diameter. Droplets suspension was incubated on ice for ~1h, then 5 min at 70°C and put back in ice. Emulsion was broken, beads washed and harvested and hybridized RNA was reverse transcribed as described in Macosko et al., 2015. For each sample, PCR amplification with 17 cycles was performed on populations of ~32000 beads (∼1600 STAMPs [single-cell transcriptomes attached to microparticles]) divided in ~4000 beads per well on eight-wells PCR strips. Size distribution and concentration were measured using Agilent Bioanalyzer (High Sensitivity 2100). Single-cell sequencing libraries were generated by tagmentation using Nextera XT (Illumina, Cat# FC-131-1024) and size distribution and concentration were measured again with Agilent Bioanalyzer (High Sensitivity 2100) and Qubit Fluorometric Quantitation respectively. Sequencing was performed on the Illumina NextSeq500 sequencer with a loading concentration of 1.55 pM, paired ends with 20 bp for read 1 and 50 bp for read 2. Paired-end sequence reads were processed as described in Mackosko et al., 2015, with the Java tools provided. After de-multiplexing, bases 1-12 (barcode) and 13-20 (unique molecular identifier; UMI) from read 1 were used to identify the cell of origin and molecule (transcript) of origin, respectively. Low quality read pairs with a cell barcode or UMI base quality less than 10 were filtered out. Read 2 was trimmed at the 5' end to remove the SMART adaptor sequence, and at the 3' end to remove polyA tails. Sequences were then aligned to the human genome (GRCh38.88) using HISAT2 v2.1.0 (Kim D, Langmead B and Salzberg SL., Nature Methods 2015) with default settings.   Reads uniquely mapped to exonic regions of genes were recorded and grouped by cell barcode. Correction for barcode synthesis errors was performed as previously described (Shekhar et al., 2016). A digital expression matrix containing the number of UMIs in each gene within each cell was then assembled, with UMIs within an edit distance of 1 collapsed. Digital expression matrices were loaded in R::Seurat and doublets and cells with low number of UMIs were removed (Germain P (2021). scDblFinder: scDblFinder)