Extended Data Fig. 2: Effects of salts, PEG and additives on Smart-seq3 reverse transcription. | Nature Biotechnology

Extended Data Fig. 2: Effects of salts, PEG and additives on Smart-seq3 reverse transcription.

From: Single-cell RNA counting at allele and isoform resolution using Smart-seq3

Extended Data Fig. 2

(a) Testing the performance of Maxima H-minus reverse transcription reactions on different reaction conditions. For each condition, we summarized boxplots with the number of unique UMIs detected in individual HEK293FT cells at 1 M raw fastq reads. We tested reverse transcription in either the standard KCl based buffer or using NaCl or CsCl. Moreover, we evaluated the effects of adding of 5% PEG or 1 mM dCTP (n = 16 cells per condition). (b) Reaction conditions as in (a) summarized against the number of genes identified from 1 million raw UMI-reads per cell (n = 16 cells per condition). (c) Reaction conditions as in (a) summarized against the number of genes identified from 1 million raw reads (sub-sampling from both 5’ UMI and internal reads) per cell (n = 16 cells per condition). (d) Genes were classified as having improved detection in Na or Cs salt (either going from undetected to detected, or positive log2 FC > 2 in UMI counts) versus detection in K salt buffer (n = 16 cells per condition). Boxplots show GC content in unchanged genes (n = 9,686), and genes with improved detection in Na (n = 8,477) and Cs salts (n = 6,261). Significance in GC content of gene sets were evaluated using two-sided t-tests, as indicated in figure. Boxplots denote median and first and third quartiles. Whiskers indicate the most extreme data point within 1.5 lengths of the box. (e) Genes with improved detection in Na and Cs are highly consistent. Shown are log2 fold-changes for genes when compared UMI counts in Na and Cs salt buffer compared to K salt buffer.

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