GSE109535
scFTD-seq: freeze-thaw lysis based, portable approach toward highly distributed single-cell 3' mRNA profiling
We present sc-FTDseq, a microchip platform for single-cell freeze-thaw lysis directly toward 3' mRNA sequencing. It offers format flexibility with a much simplified, widely adoptable workflow that reduces the number of preparation steps and hands-on time, with the quality of data and the cost per sample matching that of the state-of-the-art scRNA-seq platforms. Freeze-thaw, known as an unfavorable lysis method resulting in RNA fragmentation, turns out to be fully compatible with single-cell 3' mRNA sequencing, which detects only ~50 bases at the 3' end. We applied it to the profiling of mixed populations including whole tumors for distinguishing all major cell types and to the profiling of circulating follicular helper T cells implicated in systemic lupus erythematosus pathogenesis. Our results delineate the heterogeneity in the transcriptional programs and effector functions of these rare pathogenic T cells. Overall design: mRNA sequencing libraries are prepared after lysing the cells using three cycles of freeze-thaw. Open-surface and closed-environment formats refer to how a microwell array is used for co-loading cells and uniquely barcoded beads for mRNA capture. In closed-environment format, the microwell array is bonded with a channel on top of the array, and the cells and beads are introduced into the microwells through the channel. In open-surface format, microwell arrays are used without a channel on top, and cells and beads are introduced onto the array using a pipette.