Seq-Well: A Sample-Efficient, Portable Picowell Platform for Massively Parallel Single-Cell RNA Sequencing

Abstract

Seq-Well is a low-cost picowell platform that can be used to simultaneously profile the transcriptomes of thousands of cells from diverse, low input clinical samples. In Seq-Well, uniquely barcoded mRNA capture beads and cells are co-confined in picowells that are sealed using a semipermeable membrane, enabling efficient cell lysis and mRNA capture. The beads are subsequently removed and processed in parallel for sequencing, with each transcript's cell of origin determined via the unique barcodes. Due to its simplicity and portability, Seq-Well can be performed almost anywhere.

Keywords: Picowells; RNA-Seq; Seq-Well; Single-cell RNA sequencing; Single-cell genomics; Systems biology; Transcriptomics.

Fig 1.

Functionalized membranes can be stored in 1x PBS for 24 hours.

Fig 2.

Apply beads to the array in a dropwise fashion.

Fig. 3.

Create capillary flow to draw excess beads from the center of the array.

Fig 4.

Use tweezers to position the membrane on the glass slide so that there is a small overhang and touch it to the array just above the boundary of the wells.

Fig 5.

Hold the membrane firmly against the array with a clean glass slide.

Fig 6.

Slide the hand holding the membrane across the array to apply the membrane.

Fig 7.

Place the array in a clamp and heat it at 37°C for 30 minutes to seal the membrane.

Fig 8.

After placing the array in wash buffer, remove the membrane with tweezers

Fig 9.

Carefully position the array over a conical of wash buffer and pipette on the array to dislodge beads.

Fig 10.

Make sure the array faces outward so that the beads will fall out of wells during centrifugation.

Fig 11.

An ideal WTA product distribution has a peak at 900 – 1100 bps and has a long tail reaching 5000 bps.

Fig 12.

An ideal NTA product distribution is a smooth bell curve with a peak between 600 and 750 bps.