GSM8530830: CA5, GEX; Homo sapiens; RNA-Seq
Nuclei were isolated from solid tissues using a Singulator 100 system (S2 Genomics) following the manufacturer's protocol. Debris was removed using a Percoll gradient. Nuclei concentration was determined using a LUNA-FL™ Automated Fluorescence Cell Counter (Logos Biosystems), and morphology was examined by microscopy. The isolated nuclei were then incubated in a Transposition Mix containing Transposase. Libraries were prepared using the Chromium controller (10x Genomics) according to the Chromium Single Cell Multiome ATAC + Gene Expression protocol (CG000338). Briefly, transposed nuclei were mixed with master mix and loaded with Single Cell Multiome gel beads and partitioning oil into a Chromium Chip J. The gel beads contained poly (dT) sequences for gene expression (GEX) library preparation and spacer sequences for assay for transposase-accessible chromatin (ATAC) library preparation. Gel bead-in-emulsion (GEM) incubation resulted in 10x barcoded DNA from transposed DNA (for ATAC) and 10x barcoded cDNA from polyadenylated mRNA (for GEX). After pre-amplification, the PCR product was divided for separate ATAC and GEX library construction steps. For the ATAC library, P7 adapters and sample indices were added to pre-amplified transposed DNA during library construction via PCR. For the GEX library, pre-amplified cDNA underwent PCR amplification, followed by fragmentation, end repair, A-tailing, adapter ligation, and index PCR. The purified libraries were quantified using qPCR according to the qPCR Quantification Protocol Guide (KAPA Biosystems). Library quality was assessed using an Agilent Technologies 4200 TapeStation (Agilent technologies). Sequencing was performed on a HiSeq platform (Illumina) following the read length in the user guide. OTHER: snMultiome-seq