GSM9571677: Multiome - ATAC - CTPI2; Mus musculus; ATAC-seq
Approximately 25 mg of frozen tissue from each pooled sample was used for nuclei isolation. The nuclei isolation was performed at 4℃ using a Singulator 200 instrument (S2 Genomics) with a regular cartridge, according to the manufacturer instructions. The Single Shot Extended Nuclei Isolation program was used with a 10 min incubation time and the mixing type modified for trituration. Nuclei isolation was performed in the presence of 1U/ul of RNase inhibitor (Roche). After incubation, the samples were passed through a 40 µm Flowmi cell strainer (Electron Microscopy Sciences), and the flow through was then centrifuged at 500 g for 5 mins at 4℃. After centrifugation, the nuclei were resuspended in Diluted nuclei buffer. The nuclei concentration was assessed by PI staining using a Cellometer K2 cell counter. Between 8000-10000 nuclei were targeted for capture and used for single nuclei snATAC + snRNA. Single nuclei snATAC + snRNA-seq was performed using the 10x Genomics platform. Between 8000 and 10000 nuclei per sample were subjected to transposase assays before proceeding to single-cell partitioning into gel beads in emulsion, barcoding, and pre-amplification according to the manufacturer instructions. ATAC library construction and cDNA, followed by GEX library construction, were also done following established 10x Genomics protocols. The libraries' concentration was measured using Qubit High Sensitivity assays (Thermo Fisher Scientific), and library profiles were assessed in a fragment analyzer (Agilent) before sequencing. The snATAC and snRNA libraries were sequenced for 50bp and 100 bp paired end sequencing, respectively, on a NovaSeq X 10B instrument (Illumina) before demultiplexing and alignment to the reference mouse genome