GSM7596753: NPPO-6-H3.3K36M, multiome snRNA-seq; Mus musculus; OTHER
For scRNA-seq, organoids were dissociated by incubation with pre-warmed TrypLE at 37°C for 10 min, neutralization with 1:10 5% CS-FBS/PBS, centrifugation at 1000 rpm for 1 min, and filtration three times through a 40 µM cell strainer (Corning 431750). Cells were spun at 1000 rpm for 5 min, pellets were dissociated with 5% CS-FBS/PBS, and resuspended at 1000 cells/µl after counting using a Countess II FL Automated Cell Counter. Approximately 5,000 cells were loaded onto the Chromium Controller (10X Genomics). For multiome snATAC-seq + snRNA-seq, organoids were digested into single cells using TrypLE, passed through a 40 µM cell strainer three times, and cell numbers were quantified using an automated cell counter. Approximately 1 million cells in 0.04% BSA were used for single nuclei isolation. Cells were spun at 1000 rpm for 5 min at 4°C, dissociated for 5 min with 100 µl ice-cold lysis buffer, neutralized with 1 ml wash buffer, spun at 1000 rpm for 5 min at 4°C, and resuspended in chilled Nuclei buffer included in the Single Cell Multiome ATAC Kit (10X Genomics). Wash buffer contained 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 1% BAS, 0.1% Tween-20, 1 mM DTT (Millipore-Sigma 646563), 1 U/µl RNase inhibitor (Millipore-Sigma 3335399001) in nuclease-free water. Lysis buffer contained 10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl2, 1% BAS, 0.1% Tween-20, 0.1% Nonidet P40 Substitute, 0.01% Digitonin, 1 mM DTT, 1 U/µl RNase inhibitor in nuclease-free water. Nuclei concentration was determined using a Countess II FL Automated Cell Counter. Approximately 5,000 nuclei were loaded onto the Chromium Controller. scRNA-seq libraries were prepared following the manufacturer's instructions (Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.1, 10X Genomics). Briefly, cells were loaded onto the Chromium Controller (10X Genomics) to generate Gel Beads-in-emulsion (GEMs), and barcoded, full-length cDNA from poly-adenylated mRNA was generated and amplified via PCR. scRNA-seq libraries were prepared for paired-end sequencing, and data were processed with Cell Ranger software (10x Genomics, version 2.1) by the Columbia University Single Cell Analysis Core. Multiome snATAC+snRNA-seq libraries were prepared following the manufacturer's instructions (Chromium Next GEM Single Cell Multiome Reagent Kit,10X Genomics). Briefly, nuclei suspensions are transposed and adapters were added to the ends of the DNA fragments. Single Cell Multiome ATAC + GEX Gel Beads include a poly(dT) sequence that enables production of barcoded, full-length cDNA from poly-adenylated mRNA for gene expression (GEX) library and a Spacer sequence that enables barcode attachment to transposed DNA fragments for ATAC library. The GEMs are generated by combining barcoded Gel Beads, transposed nuclei, and a Master Mix. Barcoded transposed DNA and barcoded full-length cDNA from poly-adenylated mRNA are amplified via PCR. Libraries were prepared for paired-end sequencing, and data were processed with Cell Ranger ARC software (10x Genomics, version v2.0.2) by the Columbia University Single Cell Analysis Core.