Extended Data Fig. 3: Detailed assay design and performance metrics of scifi-RNA-seq.
a, Schematic outline of scifi-RNA-seq including detailed oligonucleotide sequences. The reverse transcription is performed inside permeabilized cells or nuclei on a 96-well or 384-well plate, introducing well-specific round1 barcodes into the whole transcriptome. Pre-indexed cells or nuclei are pooled and encapsulated into emulsion droplets using a standard microfluidic droplet generator (10x Genomics Chromium). The round2 barcodes are introduced by thermocycling ligation with a complementary bridge oligo and thermostable ligase. The droplet emulsion is then broken, and a second defined end is introduced into the library via template switching. cDNA is enriched and tagmented with a custom i7-only transposome. Finally, the library is PCR-enriched, with the option to introduce an additional sample index. The read structure for next-generation sequencing on the Illumina NovaSeq 6000 and NextSeq 500 platforms is shown. b, Nuclei recovery after pre-indexing of the whole transcriptome by reverse transcription. scifi-RNA-seq achieves high recovery rates for both cell lines and primary material. c, Nuclei with pre-indexed transcriptome, prior to microfluidic device loading, visualized under a microscope in a counting chamber. The selected image (representative of two replicate samples) shows nuclei derived from human primary T cells. d, Typical size distribution of enriched cDNA obtained with scifi-RNA-seq. e, Typical size distribution of final scifi-RNA-seq libraries ready for next-generation sequencing. f, Distribution of DNA bases along scifi-RNA-seq sequencing reads, showing the characteristic sequence patterns of the UMI, round1 barcode, sample barcode, round2 barcode, and transcript. g, Heatmap showing sequencing quality (Qscore) for each sequencing cycle.
