GSM8097133: SIGNAL-seq ADT library of CRC PDO and CAF co-cultures sublibrary 76; Homo sapiens; OTHER
PDOs were extracted from matrigel with ice cold PBS. PDOs were then digested to a single-cell suspension with TrypLE Express Enzyme (Gibco #12604013) for 10 minutes at 37C on a heated orbital shaker at 300rpm. Cells were then re-suspended in 1 mL PBS + RNAse inhibitor cocktail (RI). Fixation and permeabilisation was then carried out with a modified SPLiT-seq protocol. Single-cell suspensions were fixed with on ice in 1% effective Formaldehyde for 10 minutes before the cells were permeabilized for a further 3 minutes with 0.2% effective Triton X-100. Fixation was then quenched with 50 mM of Tris-HCL (Invitrogen #15568025) before the cells were re-suspended in 300 μL of 0.5x PBS +RI +5% DMSO before freezing and storage at -80C until processing for SIGNAL-seq. scRNA-seq RNA and ADT libraries were generated using a modified combinatorial barcoding (Rosenberg and Rocco et al., 2018) SIGNAL-seq protocol producing two sequencing libraries (RNA and ADT) for each sublibrary. SIGNAL-seq