Smart‑seq2 Mini‑bulk: Working Range and Library Quality

Application

Synthetic RNase inhibition maintains Smart‑seq2 cDNA yield and quality comparable to protein-based RRIs while decreasing primer‑dimer formation.

Low-template, full length RNA-sequencing is highly sensitive to RNA degradation, making robust RNase inhibition essential for accurate transcriptome capture. Conventional recombinant RNase inhibitors are heat-sensitive and require reducing agents. To evaluate whether SEQURNA can fully replace protein‑based inhibitors in a full‑length protocol, its performance was systematically assessed in Smart‑seq2 mini‑bulk libraries and compared with a standard recombinant RNase inhibitor.​

Smart‑seq2 cDNA libraries were generated from 100 pg of mouse RNA using lysis buffers containing a titration of SEQURNA (0–30 U/µl, corresponding to 0–13.5 U/µl during reverse transcription) or a standard buffer with recombinant RNase inhibitor. Capillary electrophoresis revealed that SEQURNA concentrations in the range of approximately 1.5–6 U/µl produced cDNA fragment size distributions closely matching those obtained with the protein inhibitor (Fig. 1a, b). Additionally, SEQURNA decreased the proportion of short 20–50 bp fragments corresponding to primer‑dimers, increasing the share of informative library molecules (Fig. 1c).​

Defined optimal SEQURNA working range in Smart‑seq2 mini‑bulk (≈1.5–6 U/µl in lysis).​

Primer‑dimer amplification in cDNA libraries is reduced

Sequencing of these Smart‑seq2 libraries demonstrated that SEQURNA within the effective range (1.5–12 U/µl in lysis) yielded numbers of detected genes, exonic read fractions,base‑level match probabilities, and gene‑body coverage that were comparable to the recombinant inhibitor (Fig. 1g-j), with similar substitution and indel rates indicating unchanged polymerase fidelity (not shown) . Collectively, these results show that SEQURNA supports a broad operational window in Smart‑seq2 where cDNA yield and sequencing quality are maintained or enhanced relative to standard protein‑based inhibitors, while reducing primer‑dimer formation.​

cDNA yield and sequencing metrics match or exceed recombinant RNase inhibitors.​ 

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