In order to capture all types of RNA present in cells while excluding ribosomal RNA, we employed reverse transcription (RT) with a combination of two types of primers. We computationally selected a set of 220 hexamers designed to selectively enrich for all non-rRNA targets. In silico alignment of these primers on the transcriptome revealed that, for every 100 nt of total RNA, 4.4 sequences align, capturing 99.9% of total RNA, thus providing ample priming coverage. (Supplementary Figure 1.A) To enhance priming efficiency, polyTN8 primer was added to the primer mixtures. The polyTN8 primer is composed of three parts: a polyT of 12 nucleotides, followed by CCC, and ending with a random octamer. The polyT anneals to the polyA tail of mRNA, with CCC serving as a bridge so that the random octamer can anneal with upstream part of the mRNA. Since mRNA in cells naturally forms secondary structures (Georgakopoulos-Soares, Parada, and Hemberg 2022), the folded RNA would be spatially closer to the polyA tail for N8 annealing. We integrated the rDS and polyTN8 primers to single cell barcode beads with handles for library preparation as well as Unique Molecule Identifier (UMI)s for PCR duplication removal. With droplet-based microfluidics platform, we introduced sc-rDSeq as a high-throughput single-cell, full-length total RNA sequencing method. (Figure 1 A) We implemented a ramping-cycling-short RT strategy to improve the reaction efficiency. The temperature is gradually ramped from 4°C to 50°C, with a corresponding hold time determined by the melting temperature of the assigned rDS sequences, and the ramping program is repeated for 20 cycles to enhance the annealing of primers that were not initially annealed. Furthermore, to prevent strand displacement that is known to occur during RT reactions (Martín-Alonso et al. 2020), the total reaction time for the RT (50°C) is limited to 5 minutes. With all these modifications, the RT efficiency was significantly improved
