GSM9648398: RNA, Donor2, A5-A7 (Cortex)-1; Homo sapiens; RNA-Seq
Ground frozen tissue was homogenized in 9 mL lysis buffer composed of 0.32 M sucrose, 0.1% Triton X-100, 5 mM MgCl₂, 25 mM KCl, 10 mM Tris-HCl (pH 8.0), 1 mM DTT, Roche cOmplete Mini Protease Inhibitor, 20 U/mL SUPERaseIN, and 20 μg/mL RNH1. Homogenization used glass Dounce homogenizers, followed by sequential filtration through 70 μm (Corning, 352350) and 30 μm (Sysmex, 04-004-2326) strainers. Myelin debris was removed by adding a sample-specific volume of myelin removal beads (Miltenyi Biotec, 130-096-433) to the homogenate, incubating on a rotating mixer at 4°C for 15 min, and passing the suspension through LS columns (Miltenyi Biotec, 130-042-401) on a QuadroMACS Separator (Miltenyi Biotec, 130-091-051). Bead volume was adjusted per sample based on tissue myelin content. Flow-through was collected and columns were washed with 2 mL wash buffer (0.1% Triton X-100, 5 mM MgCl₂, 25 mM KCl, 10 mM Tris-HCl pH 8.0, 1 mM DTT, Roche cOmplete Mini Protease Inhibitor, 20 U/mL SUPERaseIN, and 20 μg/mL RNH1). Samples were centrifuged at 1,000g for 10 min at 4°C in a Thermo Scientific Sorvall Legend RT+ benchtop centrifuge (Thermo, 75004377) with a swinging bucket rotor (Sorvall, 75006445). Supernatant was aspirated carefully and the pellet was resuspended in 1 mL sort buffer: DPBS, 1% BSA, 1 mM EDTA, 1 mM DTT, Roche cOmplete Mini Protease Inhibitor, 20 U/mL SUPERaseIN, and 20 μg/mL RNH1. For NeuN staining and FANS sorting, Mouse anti-NeuN conjugated to Alexa Fluor 488 (Millipore, MAB377X) and Hoechst 33342 (Invitrogen, H3570) were added at 1:1000. Nuclei were incubated on a rotating mixer at 4°C for 30 min and then diluted to 750,000 nuclei/mL using additional sort buffer prior to FANS. Sorting was performed on a BD Influx cell sorter (BD Biosciences, 646500) using “1-drop pure” mode with a 100 μm nozzle and sheath pressure of 20 PSI; 1× PBS served as sheath fluid, and the nuclei were kept at 4°C. Hoechst-positive nuclei with 2N DNA content were gated using SSC-Area × Hoechst-Area. Debris was excluded using FSC-Area × SSC-Area, and aggregates were excluded using FSC and SSC pulse Area- and Width-based parameters. Nuclei were then gated by NeuN (488), and NeuN-positive and NeuN-negative fractions were collected into separate 2 mL DNA LoBind tubes (Eppendorf, 022431048) containing 250 μL 5× collection buffer (DPBS, 5% BSA, Roche cOmplete Mini Protease Inhibitor, 100 U/μL SUPERaseIN, and 100 μg/mL RNH1). Collected nuclei were centrifuged at 300g for 5 min at 4°C, followed by 500g for 10 min, using an Eppendorf 5417R microcentrifuge with a swinging bucket rotor (Eppendorf A-8-11). Supernatant was removed to ~250 μL per tube. Nuclei from matching populations were pooled and quantified. To assemble indexed pA-Tn5 complexes compatible with the Chromium Next GEM Single Cell Multiome kit (10x Genomics, 1000283), we synthesized all custom oligonucleotides at a concentration of 100 µM. Custom oligos were mixed in two separate PCR tubes (10 µL AdapterA + 10 µL pMENT-A, and 10 µL AdapterB + 10 µL pMENT-B) and annealed in a thermal cycler using the following program: 95 °C for 5 min, then slow cooling to 12 °C, at 0.1 °C/s. The 20 µL annealed oligos were mixed with 120 µL unloaded pA-Tn5 (0.5 mg/mL, MacroLab) in each tube and incubated at room temperature for 30 mins, followed by an additional 10 mins at 4 °C. Equal volumes of assembled pA-Tn5-AdapterA/pMENT-A and pA-Tn5-AdapterB/pMENT-B were then combined to form functional indexed pA-Tn5 complexes, which were stored at -20 °C. Sorted NeuN⁺ and NeuN⁻ nuclei were pelleted using a swinging-bucket centrifuge (5804R, Eppendorf) and counted using a cell counter (RWD C100-Pro) with DAPI staining. For nuclei extracted from the same tissue block, we aimed to obtain 0.5 million nuclei per histone mark. For cortical regions and pons, NeuN⁺ and NeuN⁻ nuclei were combined to achieve ~50% of NeuN⁺ nuclei. For cerebellum, NeuN⁺ and NeuN⁻ nuclei were combined to achieve ~50% of NeuN⁻ nuclei. In this study, we used antibodies against histone modification marks that performed well in prior studies: H3K27ac (Abcam, ab4729) and H3K27me3 (Abcam, ab192985). Indexed pA-Tn5-antibody conjugates were assembled by mixing 1 µg antibody with 1 µL assembled pA-Tn5 (0.4 mg/mL) in 30 µL MED#1 gently, rotating for 1 hr at room temperature, and then adding 45 µL MED#1. In parallel, 0.5 million nuclei per tube were permeabilized with OMNI buffer on ice for 4 min and washed once with high-salt MED#1 buffer. Permeabilized nuclei were resuspended in 75 µL of the conjugate-containing mixture and incubated at 4 °C overnight for epitope targeting. Indexed pA-Tn5 and antibodies were removed by washing twice with MED#2 buffer. Tagmentation was performed in 70 µL MED#2 buffer supplemented with 10 mM MgCl₂ (Invitrogen, AM9530G) at 37 °C for 1 h at 550 r.p.m. in a ThermoMixer (Eppendorf), and then the reaction was terminated by adding 2× stop solution. Nuclei were washed once with 1× nuclei buffer, concentrated within 20 µL 1× nuclei buffer, and counted again using DAPI. Nuclei tagmented with different indexed pA-Tn5 complexes were combined in equal proportions, and 25,000-30,000 nuclei were aliquoted and loaded for a single droplet-generation reaction using the Chromium Next GEM Single Cell Multiome kit. DNA and RNA library amplification was then performed according to the Chromium Next GEM Single Cell Multiome kit manual with increased amplification cycles for the DNA library. Final libraries were sequenced on a NovaSeq (Illumina) platform using standard Illumina primers. For DNA libraries, read lengths were 100 + 8 + 27 + 100 (Read 1 + Index 1 + Index 2 + Read 2). For RNA libraries, read lengths were 28 + 10 + 10 + 90 (Read 1 + Index 1 + Index 2 + Read 2)