Lower limit of starting cDNA for tagmentation libraries. (A,B) Bioanalyzer electropherograms of tagmented DNA from reactions using 500 pg to 0.1 pg cDNA using either Nextera XT (A) or in-house Tn5 and reaction conditions including TAPS buffer and 8% PEG 8000 (B). (C) Technical variability in transcriptome libraries generated with Nextera XT or in-house buffers (std, standard Tn5 amounts; low, 1/100th Tn5 amount). Mean and standard deviation in gene expression estimates within replicates per protocol and in bins of genes sorted according to expression levels. Error bars, SEM (n ≥ 3). (D) Sensitivity in transcriptome libraries generated with Nextera XT or in-house buffers (std, standard Tn5 amounts; low, 1/100th Tn5 amount). We report the mean percentage of genes reproducibly detected in replicate experiments (all pairwise comparisons per protocol) and binned according to expression level, with 90% confidence intervals.
