Supplementary Figure 2: Seq-Well Experimental Workflow (see Seq-Well Protocol – Ref. 21)
From: Seq-Well: portable, low-cost RNA sequencing of single cells at high throughput
Cells are obtained from complex tissues or clinical biopsies, and digested to form a single-cell suspension. Barcoded mRNA capture beads are added to the surface of the microwell device, settling into wells by gravity, and then a single-cell suspension is applied. The device is sealed using a semi-permeable membrane that, upon addition of a chemical lysis buffer, confines cellular mRNAs within wells while allowing efficient buffer exchange. Liberated cellular transcripts hybridize to the bead-bound barcoded poly(dT) primers that contain a cell barcode (shared by all probes on the same bead but different between beads) and a unique molecular identifier (UMI) for each transcript molecule. After hybridization, the beads are removed from the array and bulk reverse transcription is performed to generate single-cell cDNAs attached to beads. Libraries are then made by a combination of PCR and tagmentation, and sequenced. After, ssingle-cell transcriptomes are assembled in silico using cell barcodes and UMIs.
