Journal: Nature Biotechnology

Article Title: Fast and highly sensitive full-length single-cell RNA sequencing using FLASH-seq

doi: 10.1038/s41587-022-01312-3

Figure Lengend Snippet: a , Estimated protocol duration to process a 96-well plate of HEK293T cells for the full-length scRNA-seq protocols used in this study. Steps are color-coded. QCs include concentration and size distribution measurements. SSsc, SMART-Seq Single Cell Kit (Takara). b , Number of genes detected in HEK293T cells processed with SS2 ( n = 80), SS3 ( n = 51) and FS ( n = 105) at two read thresholds, with reads downsampled to 500,000 (= 500K) raw reads. c , Mean ± standard deviation (s.d.) gene-body coverage in HEK293T cells. d , Cell-to-cell Kendall’s tau correlations among gene expression in SS2 ( n = 80), SS3 ( n = 51) and FS ( n = 105) using only genes expressed in all three methods ( n genes = 20,042). e , FS-LA workflow. The number of required PCR cycles is a function of the cell RNA content. f , Number of genes detected in HEK293T cells processed with FS ( n FS-19c = 56) or FS-LA ( n FS-LA-12c = 31, n FS-LA-10c = 32, n FS-LA-8c = 31, n FS-LA-6c = 24, n FS-LA-4c = 32) using 250 K downsampled raw reads. Gene detection threshold was set to >0 or >5 reads. g , Top panel shows the percentage of read tags mapped to exonic (= CDS exons), intronic or intergenic features in HEK293T cells processed with FS or FS-LA, measured using ReSQC. Bottom panel shows mapping statistics with the percentage of uniquely mapped, multimapped or unmapped reads for FS and FS-LA. h , Mean ± s.d. number of detected genes (>0 reads) per cell type in hPBMC samples. Only points supported by two or more cells are displayed. Some cells had insufficient coverage to be represented at each point. The difference between the number of genes in FS and FS-LA was evaluated at 125 K reads for each cell type using a Wilcoxon rank-sum test (two-sided, Bonferroni correction, adjusted P value). No statistically significant differences (NS) were observed ( P > 0.05). MAIT, mucosal-associated invariant T cell; mono, monocytes; NK, natural killer; T CM , central memory T cell; T EM , effector memory T cell; gdT, gamma-delta T-cell; pDC, plasmacytoid dendritic cell; cDC2, conventional dendritic cell 2. A two-sided Dunn’s test was used, and Bonferroni corrected, adjusted P values are shown for b , d and f . Box plots in f and g show the median (center), 25th/75th percentile (lower/upper hinges), 1.5× interquartile range (whiskers) and outliers (points).

Article Snippet: SSsc, SMART-Seq Single Cell Kit (Takara). b , Number of genes detected in HEK293T cells processed with SS2 ( n = 80), SS3 ( n = 51) and FS ( n = 105) at two read thresholds, with reads downsampled to 500,000 (= 500K) raw reads. c , Mean ± standard deviation (s.d.) gene-body coverage in HEK293T cells. d , Cell-to-cell Kendall’s tau correlations among gene expression in SS2 ( n = 80), SS3 ( n = 51) and FS ( n = 105) using only genes expressed in all three methods ( n genes = 20,042). e , FS-LA workflow.

Techniques: Concentration Assay, Standard Deviation, Expressing

Journal: Journal of Biomolecular Techniques : JBT

Article Title: Benchmarking Single-Cell mRNA–Sequencing Technologies Uncovers Differences in Sensitivity and Reproducibility in Cell Types With Low RNA Content

doi: 10.7171/3fc1f5fe.dbeabb2a

Figure Lengend Snippet: Testing overview for SSsc and SSlp kits. For all testing schema, sample preparation was followed by library preparation, Illumina sequencing, and analysis using Cogent AP software. (A) Workflow for testing cultured cells (GM12878 and CHO cells) isolated by Fluorescence-activated cell sorting (FACS). This workflow was used for a performance comparison between SSsc and SS2 and for verifying compatibility with miniaturized volumes on the MANTIS (Formulatrix) and mosquito (SPT Labtech) (Figs. 2 and 5). (B) Workflow for performance comparison between SSsc and SS3. CD3+ T cells were isolated from human PBMCs by FACS, and the appropriate user manual or protocol was followed for RNA isolation (Fig. 3). (C) Workflow for performance comparison between SSsc PLUS (SSsc + SSlp) and Nextera XT using isolated RNA (control mouse brain RNA; Fig. 4).

Article Snippet: Outliers are plotted as empty circles. ( C ) Gene counts for transcripts per million (TPM) > 0.1 with SSsc PLUS compared with Nextera XT [ n = 6; medians = 14,643 (SSlp) and 14,494 (Nextera XT)]. ( D ) Representative x - y plots of 2 example libraries from the same cDNA prepared with SSsc PLUS or Nextera XT in duplicate and the same cDNA library compared between Nextera XT and PLUS.

Techniques: Sample Prep, Illumina Sequencing, Software, Cell Culture, Isolation, Fluorescence, FACS, Comparison, Control

Journal: Journal of Biomolecular Techniques : JBT

Article Title: Benchmarking Single-Cell mRNA–Sequencing Technologies Uncovers Differences in Sensitivity and Reproducibility in Cell Types With Low RNA Content

doi: 10.7171/3fc1f5fe.dbeabb2a

Figure Lengend Snippet: Comparing library preparation of Nextera XT and SSsc PLUS. A) Higher library yields for SSsc PLUS (n = 6 for each; median = 10.5 and 55.4 nM, respectively). The boxes denote the interquartile range (IQR) (i.e., the 25th and 75th quartiles); the whiskers are 1.5´ IQR from the median value and represent the extremes of the data. Outliers are plotted as empty circles. (B) Comparable distribution of reads for major genomic categories (i.e., mitochondria, rRNA, intergenic, intronic, and exonic; n = 6). The boxes denote the IQR (i.e., the 25th and 75th quartiles); the whiskers are 1.5´ IQR from the median value and represent the extremes of the data. Outliers are plotted as empty circles. (C) Gene counts for transcripts per million (TPM) > 0.1 with SSsc PLUS compared with Nextera XT [n = 6; medians = 14,643 (SSlp) and 14,494 (Nextera XT)]. (D) Representative x-y plots of 2 example libraries from the same cDNA prepared with SSsc PLUS or Nextera XT in duplicate and the same cDNA library compared between Nextera XT and PLUS. R2 values are shown in the graph; Nextera XT R2 = 0.9961, PLUS R2 = 0.9978, and Nextera XT versus PLUS R2 = 0.9579.

Article Snippet: Outliers are plotted as empty circles. ( C ) Gene counts for transcripts per million (TPM) > 0.1 with SSsc PLUS compared with Nextera XT [ n = 6; medians = 14,643 (SSlp) and 14,494 (Nextera XT)]. ( D ) Representative x - y plots of 2 example libraries from the same cDNA prepared with SSsc PLUS or Nextera XT in duplicate and the same cDNA library compared between Nextera XT and PLUS.

Techniques: cDNA Library Assay

Journal: Journal of Biomolecular Techniques : JBT

Article Title: Benchmarking Single-Cell mRNA–Sequencing Technologies Uncovers Differences in Sensitivity and Reproducibility in Cell Types With Low RNA Content

doi: 10.7171/3fc1f5fe.dbeabb2a

Figure Lengend Snippet: Testing overview for SSsc and SSlp kits. For all testing schema, sample preparation was followed by library preparation, Illumina sequencing, and analysis using Cogent AP software. (A) Workflow for testing cultured cells (GM12878 and CHO cells) isolated by Fluorescence-activated cell sorting (FACS). This workflow was used for a performance comparison between SSsc and SS2 and for verifying compatibility with miniaturized volumes on the MANTIS (Formulatrix) and mosquito (SPT Labtech) (Figs. 2 and 5). (B) Workflow for performance comparison between SSsc and SS3. CD3+ T cells were isolated from human PBMCs by FACS, and the appropriate user manual or protocol was followed for RNA isolation (Fig. 3). (C) Workflow for performance comparison between SSsc PLUS (SSsc + SSlp) and Nextera XT using isolated RNA (control mouse brain RNA; Fig. 4).

Article Snippet: To compare reproducibility of library preparation between different samples using SSlp or Nextera XT, triplicate cDNA were first generated using SSsc from 10 pg of mouse brain RNA ( C ).

Techniques: Sample Prep, Illumina Sequencing, Software, Cell Culture, Isolation, Fluorescence, FACS, Comparison, Control

Journal: Journal of Biomolecular Techniques : JBT

Article Title: Benchmarking Single-Cell mRNA–Sequencing Technologies Uncovers Differences in Sensitivity and Reproducibility in Cell Types With Low RNA Content

doi: 10.7171/3fc1f5fe.dbeabb2a

Figure Lengend Snippet: Comparing library preparation of Nextera XT and SSsc PLUS. A) Higher library yields for SSsc PLUS (n = 6 for each; median = 10.5 and 55.4 nM, respectively). The boxes denote the interquartile range (IQR) (i.e., the 25th and 75th quartiles); the whiskers are 1.5´ IQR from the median value and represent the extremes of the data. Outliers are plotted as empty circles. (B) Comparable distribution of reads for major genomic categories (i.e., mitochondria, rRNA, intergenic, intronic, and exonic; n = 6). The boxes denote the IQR (i.e., the 25th and 75th quartiles); the whiskers are 1.5´ IQR from the median value and represent the extremes of the data. Outliers are plotted as empty circles. (C) Gene counts for transcripts per million (TPM) > 0.1 with SSsc PLUS compared with Nextera XT [n = 6; medians = 14,643 (SSlp) and 14,494 (Nextera XT)]. (D) Representative x-y plots of 2 example libraries from the same cDNA prepared with SSsc PLUS or Nextera XT in duplicate and the same cDNA library compared between Nextera XT and PLUS. R2 values are shown in the graph; Nextera XT R2 = 0.9961, PLUS R2 = 0.9978, and Nextera XT versus PLUS R2 = 0.9579.

Article Snippet: To compare reproducibility of library preparation between different samples using SSlp or Nextera XT, triplicate cDNA were first generated using SSsc from 10 pg of mouse brain RNA ( C ).

Techniques: cDNA Library Assay