Fig. 2
Workflow and depletion of 16S sequence with DASH. A Schematic of DASH protocol. The orange box highlights the DASH-specific steps, and all other steps are in the standard 10X protocol. Probes containing poly(T)VN, where V is any base except T, and N is any base, are used for poly(A) enrichment. cDNA is reverse-transcribed in the 10X Chromium Controller and amplified by cDNA primers, Read1 and template switching oligo (TSO). After generation of cDNA, 16S is depleted by incubation with Cas9 and sgRNAs targeting the 16S sequence, followed by post-CRISPR amplification with the same cDNA primers. The re-amplified cDNA is then fragmented and indexed for subsequent sequencing. B Schematic of experimental design to benchmark the performance of DASH. Three biological replicates of stem cells (X1 cells) are sorted and processed for cDNA preparation. cDNA is then split into “untreated” and “DASHed” libraries. Sequencing reads are processed by CellRanger and then the cell barcodes recovered from both groups are used for downstream analysis. C Venn diagram of cell barcodes from untreated and DASHed libraries. The shared cell barcodes are used to assess library quality. D Fragment analysis of untreated cDNA (top) and DASHed cDNA (bottom). x-axis shows fragment size in base pairs (bp), and y-axis shows relative fluorescence units (RFU). Red peak marks the lower marker (1 bp) and blue peak marks the upper marker (6000 bp). E Volcano plot of differential expression analysis of DASHed versus untreated 10X scRNA-seq datasets. Data includes 3 biological replicates of scRNA-seq libraries combined and analyzed as pseudobulk samples. x-axis is the average log2 fold change across all cells. y-axis is -log10 of adjusted p-value of Wilcoxon Rank Sum test based on Bonferroni correction. The cutoff for significant genes is adjusted p-value < 0.05 and absolute average log2 fold change > 1. F Volcano plot of differential expression analysis of DASHed versus untreated bulk RNA-seq datasets. Data includes 3 biological replicates of bulk RNA-seq libraries for either untreated or DASHed. x-axis is the average log2 fold change across all cells. y-axis is -log10 of adjusted p-value of Wald test based on Benjamini–Hochberg correction. The cutoff for significant genes is adjusted p-value < 0.05 and absolute average log2 fold change > 1