Fig. 2: TAS-Seq effectively suppresses excessive elongation of primer-derived by-products under various TdT reaction conditions.
From: TAS-Seq is a robust and sensitive amplification method for bead-based scRNA-seq
a and c TAS-Seq tolerance against TdT reaction time and TdT activity. TdT reaction with ddCTP:dCTP (1:20) or dCTP only with Co2+ supplementation was performed from 5 or 30 or 45 min with different TdT enzyme amounts against exonuclease I-treated BD Rhapsody beads (a) or cDNA-synthesized, exonuclease I-treated BD Rhapsody beads (c). Size distributions of the 1st PCR products were analyzed. Note that the length of primer-derived bi-products (arrows) peaked at around 136 bp and did not extend over 200 bp in every reaction time. In addition, amplified cDNA was also visible (c, arrowheads). b Quartz-seq2 tolerance against TdT reaction warming and enzyme activity. Size distribution of remained primer-derived by-products of Quartz-seq2 under standard conditions, and under pre-incubation at 23 °C for 5, 10 min, and 15; 1.5× TdT amount shown. Reactions were performed without RNA. Note that primer-derived by-products were extended over 200 bp with 23 °C pre-incubation. d Size distribution of TAS-Seq amplified cDNA library of 6000 single cells derived from the murine lung. a–d Representative results of two independent experiments are shown.
