GSM8453994: UP-10072, UP-9121, UP-9096, 1h ACh variations; Homo sapiens; RNA-Seq
GBOs were reaggregated into organoids of 2000 cells in 96-well U-bottomed plates. For ACh experiments, ACh (acetylcholine chloride, Sigma Aldrich, A6625) was applied to a final concentration of 1 mM for various durations prior to removal of medium and sample lysis. If any condition required a waiting period prior to lysis, GBOs were washed once with GBO medium prior to complete replacement with fresh GBO medium. For sequencing of CHRM3 knockdown GBOs, GBO cells were directly infected with either scrambled or knockdown lentivirus prior to reaggregation as described above for retroviral infection, and cells were subjected to 1-hour ACh treatment 7 days following shRNA transduction. Sample lysis was performed by adding 30 μL of SMART-seq3 lysis buffer (5 μL PEG 8000 (40% solution, Sigma Aldrich, P1458), 0.3 μL Triton X-100 (10% solution), 0.2 μL of 100 μM Oligo-dT30VN, 2 μL of 10 mM dNTPs (Roche, 50-196-5273), 0.2 μL Protector RNase inhibitor (Sigma Aldrich, 3335402001), 0.2 μL recombinant RNase inhibitor (Takara, 2313B) and 22.1 μL nuclease-free water (Thermo Fisher Scientific, AM9932)) to each well. For library preparation, the plate was heated to 72°C for 10 minutes, and subsequently kept at 4°C for cell lysis. 30 μL of RT mix, containing 1 μL of 1 M Tris-HCl pH 8.3 (Hampton Research, HR2-900-14), 0.24 μL of 5 M NaCl (Thermo Fisher Scientific, AM9759), 0.1 μL of 1 M MgCl2 (Thermo Fisher Scientific, AM9530G), 0.4 μL of 1 mM GTP (Thermo Fisher Scientific, R1461), 3.2 μL of 100 mM DTT (Millipore Sigma, 3483-12-3), 0.16 μL of nuclease-free water, 0.25 μL Protector RNase inhibitor (Millipore Sigma, C852A14), 0.25 μL recombinant RNase inhibitor (Takara, 2313B), 0.4 μL reverse transcriptase (Thermo Fisher Scientific, EP0753), and 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 20 μL 5X KAPA HiFi HotStart Buffer (Roche, KK2502), 3 μL of 10 mM dNTPs, 0.05 μL of 1 M MgCl2, 0.1 μL of 1mM PCR forward primer, 0.1 μL of 100 μM PCR reverse primer, 34.75 μL of nuclease-free water, and 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, 16 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 100 μL of nuclease-free water. cDNA was quantified using Qubit dsDNA HS assay kit (Life Technologies, Q32851). Each sample was then tagmented by mixing 10 μL of 0.5 ng/μL cDNA, 20 μL of 2X TD buffer (20 mM Tris pH 8.0, 10 mM MgCl2, 20% dimethylformamide (Sigma, D4551)), and 10 μL of diluted Tn5 (Thermo Fisher Scientific, TNP92110) and incubating at 55°C for 20 minutes with reaction termination upon addition of 10 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 5 μL of 10 μM Nextera i7 primer, 5 μL 10 μM Nextera i5 primer, 25 μL of 5X Phusion Plus Buffer (Thermo Fisher Scientific, F630L), 2.5 μL of 10 mM dNTPs, 2.5 μL of 1% Tween-20, 17 μL nuclease-free water, and 1 μL of Phusion Plus polymerase (Thermo Fisher Scientific, F630L). Tagmentation PCR was performed with the following thermocycling conditions: 98°C for 3 minutes, 10 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  a NovaSeq 6000 (Illumina) at a final depth of roughly 12-15M reads/sample (with one sample being one well/barcode).