GSM8499446: Sample_30_AD; Homo sapiens; RNA-Seq
We isolated nuclei from fresh-frozen brain tissue blocks obtained from the ALSP donor (white and gray matter from prefrontal cortex, and white matter from medial temporal lobe, parietal cortex, and occipital cortex), and controls (white and gray matter from prefrontal cortex). The gray or white matter was dissected under a stereomicroscope, and then chopped it into small pieces (< 1 mm³) with a chilled razor blade. For tissue homogenization, a Kimble Kontes all-glass tissue grinder was utilized (Kimble, 885300-0007). Each tissue sample was dissociated using 2.4 mL of homogenization buffer containing 10 mM Tris pH 8, 5 mM MgCl2, 25 mM KCl, 250 mM sucrose, 1 μM DTT, 0.5x protease inhibitor (cOmplete, Roche #4693159001), 0.2 U/μL RNase inhibitor, and 0.1% Triton X-100. The homogenates were subsequently filtered through a 40-μm cell strainer, transferred into 1.5-mL Eppendorf tubes, and centrifuged at 1000 ×g for 8 minutes at 4°C. The supernatant was aspirated and discarded, and the pellets were resuspended in 450 μL of cold homogenization buffer. Further clean-up was carried out using iodixanol gradient centrifugation. An equal volume (450 μL) of 50% v/v iodixanol solution (41.25 mM sucrose, 24.75 mM KCl, 4.95 mM MgCl2, 9.9 mM Tris pH 8, 50% w/v iodixanol) was added to the homogenate and gently mixed with a pipette. The mixture was then transferred to a 2-mL Eppendorf tube containing 900 μL of cold 29% iodixanol solution (129.17 mM sucrose, 77.5 mM KCl, 15.5 mM MgCl2, 31 mM Tris pH 8, and 29% w/v iodixanol) by slow layering on the top. The tubes were centrifugated at 13,500 ×g for 20 minutes at 4°C, resulting in the sedimentation of nuclei at the bottom. The supernatant and top layer of myelin and cell debris were removed and discarded. The nuclei pellets were then detached with a small amount (~50 μL) of immunostaining buffer (0.1 M PBS; pH 7.4, 0.5% bovine serum albumin [BSA], 5 mM MgCl2, 2 U/mL DNAse I, 0.2 U/μL RNase inhibitor), transferred to clean tubes, and gently resuspended in a total volume of 200 μL of immunostaining buffer. After a 15-minute incubation with immunostaining buffer at 4°C, with gentle rocking, primary antibodies (NeuN; 1:1000, Millipore cat#MAB377; and/or PAX6; 1:500, Biolegend, 901301) were added, and the suspension was further incubated for 40 minutes at 4°C with gentle rocking. Subsequently, an equal volume (500 μL) of immunostaining buffer was added, and the tubes were inverted several times before being centrifuged at 500 ×g for 5 minutes at 4°C. The supernatant was removed, and the pellets resuspended in 600 μL of immunostaining buffer. Secondary antibodies (Alexa Fluor 488, Alexa Fluor 647) and a nuclear stain (Hoechst 34580; 1:1,000 of 2.5 mg/ml stock, Invitrogen cat# H21486) were subsequently added, and the solutions incubated for 30 minutes at 4°C with gentle rocking. Microscopic evaluation of the number and morphology of the nuclei was conducted after each critical step and immediately before FANS. FANS was employed to enrich single nuclei from glia. We used a Sony SH800 cell sorter with a 100-μm microfluidics sorting chip. The primary laser, at 488 nm, was utilized for the generation of forward scatter (FSC) and back scatter (BSC). Secondary lasers emitting at 405, 488, and 638 nm were utilized to detect Hoechst 34580, Alexa Fluor 488, and Alexa Fluor 647, respectively. The FSC versus BSC gates were set with permissive limits, discarding the smallest debris and largest particles. Hoechst 34580 fluorescence was used to discriminate single nuclei from doublets and clumps. We sorted Hoechst 34580 positive singlets from ALSP white matter. For ALSP gray matter, we sorted non-neuronal (NeuN-negative) nuclei. For healthy and AD control tissue, we sorted the PAX6-positive, NeuN-negative population singlets. Nuclei were collected in collection buffer (0.1 M PBS pH 7.4, 0.1 U/μL RNase inhibitor, 1% BSA) into BSA-coated Eppendorf tubes. snRNAseq of postmortem human brain nuclei was conducted using the 10x Genomics Chromium Single Cell 3' v3 assay. The input single nuclear suspensions were centrifuged at 400 ×g for 5 min at 4°C to achieve a concentration of ~350 nuclei per μL, determined using a hemocytometer. On average, ~12,500 nuclei were loaded to capture around 5,000 nuclei per sample, with an expected capture efficiency of ~40%. cDNA amplification and library construction were performed following the manufacturer's instructions. The paired-end libraries were sequenced on Novaseq 6000. A total of 12 samples were sequenced in 3 batches. For each sequencing batch, the concentration of each sample was normalized to the total number of nuclei to ensure similar numbers of reads per nucleus. Nuclei were sequenced to a depth of ~75,000 reads per nucleus.