Omega-seq: ultra-low-background RNA sequencing with faithful molecular counting and precise transcript-end capture

PCR-based RNA sequencing generates phantom unique molecular identifiers (UMIs) when residual UMI-bearing oligonucleotides reprime during amplification, inflating molecule counts and corrupting relative abundances; non-specific amplification limits sensitivity, and synthetic poly-T tracts disrupt sequencing and obscure transcript 3' ends. This series contains the sequencing data for Omega-seq, a full-length single-cell RNA-seq method that restricts UMI incorporation to reverse transcription using uracil-containing, USER-excisable template-switching oligonucleotides together with a uracil-intolerant polymerase, and that replaces the conventional oligo-dT primer with an Omega-shaped dT primer which relocates the sequencing adapter past the homopolymer tract.

The data comprise (i) serial dilutions of human brain total RNA (10, 1 and 0.1 pg) and no-template controls processed in parallel by Omega-seq and FLASH-seq-UMI, with ERCC spike-ins, on Illumina; (ii) FACS-sorted individual Drosophila larval neuroblasts, and pooled neuroblasts and neurons, prepared by both methods; (iii) an oligonucleotide-design comparison of standard versus Omega-shaped dT primers; and (iv) a Nanopore run in which preamplified cDNA from both methods across the same dilution series was barcoded and sequenced on a single flow cell, used to characterise adapter composition and mappability of the preamplification products directly.