GSM8841694: 2d_5fu_rna; Homo sapiens; OTHER
Cells from monolayer cultures were washed with PBS and detached with trypsin for 3 min, at 37°C. PDS slices were soaked 3 times in PBS and cells were detached with Corning Accutase for 5 min at 37 °C with gentle agitation at 180 rpm. Single-cell suspensions were then generated using a 25G needle and a 40 µm cell strainer. Dead cell removal was performed using the MACS Dead Cell Removal kit (Miltenyi), according to the manufacturer's instructions. The triomics experiment was performed using the Tapestri Single-Cell Multiomics V1.1 workflow (Mission Bio). Briefly, single-cell suspensions were stained using protein-targeting AOCs and subsequently diluted to ~3500 cells/ml. Stained cells were then encapsulated in droplets by the Tapestri instrument. Subsequently, cell lysis, reverse transcription and protease treatment was run. Custom panels for DNA and RNA was used and genomic DNA, cDNA and antibody tag sequences were barcoded in emulsion. Barcoding beads were cleaved off from targeted sequences using UV light. The target sequences were preamplified after which the emulsion was broken and libraries specific for DNA, RNA and protein targets were enriched and purified using Dynabeads MyOne Streptavidin and SPRI beads. Separate libraries for each analyte were then generated by PCR using Illumina primers. Final libraries were purified using SPRI beads before quantification and quality control using capillary gel-electrophoresis on a Fragment Analyzer. Libraries were pooled and sequenced according to the manufacturer's instructions using 150 paired-end cycles, supplemented with 15% PhiX Control v3.