For closed-environment format, microfluidic devices were first infused with PBS and pressurized to remove air bubbles inside the microwells using a manually operated syringe with outlet closed. The devices were filled with 1% BSA in PBS, and incubated at room temperature for 30 min to prevent attachment of cells and molecules on PDMS surfaces. Devices were then washed with PBS prior to cell and bead loading. To establish gravity-driven flow, device outlet was connected to a 10′ tubing with a one way stopcock connected at the end while the device inlet is left unconnected to serve as a reservoir. In this configuration, solutions were simply pipetted onto the inlet reservoir and withdrawn into the device through gravity-driven flow by adjusting the height difference between the inlet and the end of the tubing connected to outlet. The one way stopcock further allowed start/stop control over the fluid flow to facilitate cell and bead loading. Similarly, the flow could also be reversed by creating a higher hydrostatic pressure on the outlet side by adjusting the height of the tubing. During scRNA-seq experiments, a single cell suspension, 5000–10 000 cells in 50 μl PBS + 1%BSA solution, was pipetted on the inlet and withdrawn into the device. Once the channel was completely filled with cell solution, the fluid flow was stopped and cells were allowed to settle by gravity. Excess cells were washed out by PBS, and mRNA capture beads, 30 000–120 000 beads in 50–150 μl, were loaded similar to cells. Size exclusion and back-and-forth loading ensured loading of >99% of the microwells with a single bead. Excess beads were washed out with PBS, and 100–200 μl freeze-thaw lysis buffer was introduced into the devices. Fluorinated oil (Fluorinert FC-40), 100–200 μl in volume, was then withdrawn into the devices to seal the microwells. After oil sealing, the tubing at the outlet was disconnected, and the microfluidic devices were exposed to three freeze thaw cycles, 5 min freezing at –80°C freezer or dry ice/ethanol bath and 5 min thawing at room temperature. Following lysis, microfluidic device was incubated for an hour inside a wet chamber for mRNA capture onto beads. mRNA binding occurs in the freeze-thaw lysis buffer without the need for buffer exchange. After incubation, the inlet of the microfluidic device was connected to a syringe filled with 6× saline-sodium citrate (SSC) buffer and the outlet was connected to eppendorf tube with a tubing. The microfluidic device was then inverted and the beads were flushed out of the device into the tube by purging. Centrifugation of the microfluidic device in inverted orientation before purging or gentle tapping on the back of the microfluidic device with a tweezer during purging was used to help move the beads out of the microwells. We were able to recover >95% of the beads using this fashion. Collected beads were centrifuged at 1000g for 1 min, and washed twice with 6× SSC buffer prior to reverse transcription.