3’ RNA sequencing for robust and low-cost gene expression profiling

Abstract 3’seq-RNA Profiling (3’SRP) approach is based on multiplexing samples and molecular indexing mRNA in order to drive genome-wide transcriptional profiling at reasonable cost in comparison to standard RNA-sequencing. The protocol is performed according to the 3′-digital gene expression (3′-DGE) approach developed by the Broad institute. The libraries are prepared from small amounts of total RNA where the mRNA poly(A) tails are tagged with universal adapters, well-specific barcodes and unique molecular identifiers (UMIs). We have improved the fragmentation step by implementing tagmentation based on the activity of a bead-linked transposome. This technique allows sample multiplexing on 96-well plates. Libraries are then sequenced using standard procedures, e.g. on Hiseq2500 or NovaSeq 6000 SP Flow Cells. We have developed a snakemake pipeline including every analysis step from raw fastq de-multiplexing to functional annotation of the differentially expressed genes, producing a complete HTML report for end-user.