PK-triggered cellular lysis and mRNA capture

Mammalian cells were stained with Calcein AM (Thermo Fisher, C3099) in 1 ml of PBS with 0.04% bovine serum albumin (BSA) according to manufacturer’s instructions. After 30 min of incubation at room temperature on a rotisserie incubator (Isotemp, Fisher Scientific), cell suspensions were quantified with a Luna-FL automated cell counter and diluted in 1× PBS with 0.04% BSA. Calcein-stained cells (1,500) in 5 µl of 1× PBS with 0.04% BSA were added to 35 µl of barcoded hydrogel templates with 29 U ml^–1 PK (NEB, P8107S) and 70 mM DTT (Sigma, D9779) and mixed for 10 pipette strokes. Care was taken to avoid generating bubbles when mixing cells with barcoded hydrogel templates. Two hundred and eighty microliters of 0.5% ionic Krytox in HFE 7500 oil⁶⁶ was added to the cell–bead mixture and vortexed at 3,000 r.p.m. for 15 s horizontally and then 2 min vertically with a custom vortexer (Fluent BioSciences, FB0002776). Oil was removed from below the emulsion such that less than 100 µl remained. The PIP emulsion was subsampled on a C-Chip disposable hemacytometer (Fisher Scientific, DHCN015) before lysis, with each subsample consisting of 3.5 µl of PIP emulsion per field of view. The C-chip was imaged in brightfield at ×2 magnification. The remaining PIP emulsion was subjected to enzymatic lysis at 65 °C for 35 min on a PCR thermocycler (Eppendorf Mastercycler Pro) with the lid temperature set to 105 °C. After lysis was complete, fluorescence images were captured using a Nikon 2000 microscope with 470-nm excitation (Thorlab, M470L5).