GSM8449599: UP-9121 and UP-9096, baseline and ACh, scRNAseq; Homo sapiens; RNA-Seq
GBOs were first dissociated into a single-cell suspension with a brain tumor dissociation kit (Miltenyi Biotech, 130-0950929). In brief, 5-10 GBOs were washed with DPBS and incubated in 1 mL of dissociation mix according to the manufacturer's protocol. GBOs were placed on a tube rotator in a 37°C incubator for 30 minutes to 1 hour with occasional pipetting to mechanically disrupt large tissue chunks. Cells were then strained through a 70 μm filter (Miltenyi Biotech, 130-110-916), centrifuged at 300g for 5 minutes and resuspended in 1 mL of GBO culture medium. Cell viability and cell concentration were measured with an automated cell counter (Thermo Fisher Scientific, Countess 3 Automated Cell Counter) with trypan blue staining (Thermo Fisher Scientific, C10312). An ideal cell viability following dissociation is typically > 80%. Cells were then resuspended in FACS pre-sort buffer (BD, 563503) with 0.2 μg/mL DAPI (BD, 564907). For sliced neocortical organoid (SNO) scRNAseq, 100-day old SNOs cultured as described above were dissociated in a similar manner as GBOs but with the addition of 50 μL Enzyme P (Miltenyi, 130-107-677) per 1 mL of dissociation mix. Single cells were sorted on a BD Influx (100 μm nozzle) with FACSDiva software into low-profile 96-well PCR plates (USA Scientific, 1402-9500) containing 3 μL of SMART-seq3 lysis buffer (0.5 μL PEG 8000 (40% solution, Sigma Aldrich, P1458), 0.03 μL Triton X-100 (10% solution), 0.02 μL of 100 μM Oligo-dT30VN, 0.2 μL of 10 mM dNTPs (Roche, 50-196-5273), 0.02 μL Protector RNase inhibitor (Sigma Aldrich, 3335402001), 0.02 μL recombinant RNase inhibitor (Takara, 2313B) and 2.21 μL nuclease-free water per reaction (Thermo Fisher Scientific, AM9932)) and 3 μL Vapor-Lock (Qiagen, 981611). Plates after sorting were briefly centrifuged, frozen on dry ice, and stored at -80°C for later processing. For library preparation, plates were thawed, heated to 72°C for 10 minutes, and subsequently kept at 4°C for cell lysis. 3 μL of RT mix, containing 0.1 μL of 1 M Tris-HCl pH 8.3 (Hampton Research, HR2-900-14), 0.024 μL of 5 M NaCl (Thermo Fisher Scientific, AM9759), 0.01 μL of 1 M MgCl2 (Thermo Fisher Scientific, AM9530G), 0.04 μL of 1 mM GTP (Thermo Fisher Scientific, R1461), 0.32 μL of 100 mM DTT (Millipore Sigma, 3483-12-3), 0.016 μL of nuclease-free water, 0.025 μL Protector RNase inhibitor (Millipore Sigma, C852A14), 0.025 μL recombinant RNase inhibitor (Takara, 2313B), 0.04 μL reverse transcriptase (Thermo Fisher Scientific, EP0753), and 0.4 μL of 20 μM TSO, was added to each well of the 96-well plate. To enable multiplexing and enhance throughput, we designed 48 unique TSOs consisting of an additional 6-bp cell barcode directly adjacent to the 5'-end of the Smart-seq3 UMI. Reverse transcription was performed with the following thermocycling conditions: 42°C for 90 minutes, 10 cycles of 50°C for 2 minutes and 42°C for 2 minutes, 85°C for 5 minutes, and 4°C indefinitely. Next, for cDNA amplification, 6 μL of PCR mix, consisting of 2 μL 5X KAPA HiFi HotStart Buffer (Roche, KK2502), 0.3 μL of 10 mM dNTPs, 0.005 μL of 1 M MgCl2, 0.01 μL of 1mM PCR forward primer, 0.01 μL of 100 μM PCR reverse primer, 3.475 μL of nuclease-free water, and 0.2 μL of KAPA HiFi HotStart polymerase (Roche, KK2502), was added to each well. cDNA amplification was performed with the following thermocycling conditions: 98°C for 3 minutes, 18 cycles of 98°C for 20 seconds then 65°C for 30 seconds then 72°C for 4 minutes, 72°C for 5 minutes, and 4°C indefinitely. cDNA from 48 wells was then pooled together into one sample (2 samples per plate), and samples were then purified twice with 0.6X then 0.8X AMPure XP beads (Thermo Fisher Scientific, A63881) and eluted in 10 μL of nuclease-free water. cDNA was quantified using Qubit dsDNA HS assay kit (Life Technologies, Q32851). Each sample was then tagmented by mixing 2 μL of 0.5 ng/μL cDNA, 4 μL of 2X TD buffer (20 mM Tris pH 8.0, 10 mM MgCl2, 20% dimethylformamide (Sigma, D4551)), and 2 μL of Tn5 (Thermo Fisher Scientific, TNP92110) and incubating at 55°C for 20 minutes with reaction termination upon addition of 2 μL of 0.2% SDS (Invitrogen, 15553-035) at room temperature for 5 minutes. We chose to use i5-only Tn5 to enrich for the 5'-ends of mRNA, although full-length sequencing can be achieved using i5/i7 Tn5. Fragments were amplified by adding 1 μL of 10 μM Nextera i7 primer, 1 μL 10 μM Nextera i5 primer, 5 μL of 5X Phusion Plus Buffer (Thermo Fisher Scientific, F630L), 0.5 μL of 10 mM dNTPs, 0.5 μL of 1% Tween-20, 6.8 μL nuclease-free water, and 0.2 μL of Phusion Plus polymerase (Thermo Fisher Scientific, F630L). Tagmentation PCR was performed with the following thermocycling conditions: 98°C for 3 minutes, 8 cycles of 98°C for 10 seconds then 60°C for 30 seconds then 72°C for seconds, 72°C for 5 minutes, and then indefinitely at 4°C. Resulting DNA was purified twice with 0.8X then 1.0X AMPure XP beads and eluted in 10 μL of 10 mM Tris, pH 8.0. Samples were then quantified by qPCR with library sizes quantified by Bioanalyzer (Agilent). Samples were sequenced on either a NovaSeq 6000 (Illumina) or NextSeq 550 (Illumina) at a final depth of roughly 100K-200K reads/cell.