Extended Data Fig. 3: Optimization of first-strand synthesis of agarose captured mRNA.
From: Identification of astrocyte regulators by nucleic acid cytometry
(a) Steric capture of cellular genomic DNA inside the agarose matrix. Left: brightfield image of agarose gels. Right: SYBR green fluorescence of stained genomes inside agarose gels. Cells are loaded at limiting dilution to ensure single-cell encapsulation; approximately one in every ten agarose hydrogels contains a cell. (b) Estimated number of cells per drop based on Poisson statistics for microfluidic loading during FIND-seq. (c) Quantification of live cells by flow cytometry using AmCyan live/dead cell dye. n = 4 mice. (d) Whole transcriptome amplification (WTA) of cDNA covalently attached to agarose beads shows full length material is captured and reverse transcribed. (e) WTA yield as a function of PCR cycle number. (f) Optimization of cDNA capture with buffer composition, enzyme, template switch oligonucleotide concentration and additives (6 mM Mg2+, 1M betaine, and 7.5% PEG-8000). (g) Quantification of the percent mitochondrial reads in bulk FIND-seq data for each replicate. n = 22 samples. (h) Calculation of score per cell from astrocytes derived from scRNA-seq from ref. 22 or each bulk FIND-seq replicate for the pathway GOBP: Execution phase of apoptosis (GO: 0097194).
