The QIAseq FX Single Cell RNA Library Kit is a complete cell-to-library solution that generates RNA-seq libraries from single eukaryotic cells or from pg-levels of purified RNA. The kit contains reagents for the efficient lysis of isolated single cells from common cell-sorting or cell-isolation platforms. After lysis, genomic DNA is degraded and reverse transcription is performed. This reaction can be primed from either an included oligo-dT primer in order to reduce the number of reads arising from ribosomal RNA and to enrich for polyadenylated RNAs, or from random primers, for example when sequencing viral RNA genomes from limited samples. After reverse transcription and second strand synthesis, a ligation step generates a long, double-stranded cDNA template which is amplified via multiple displacement annealing using a specially engineered, ultra-high fidelity phi29 polymerase. This generates µg amounts of amplified cDNA, some of which can be stored for later use or confirmatory testing. Amplified cDNA is fragmented with a highly random, enzymatic fragmentation step, which generates inserts compatible with common sequencing read lengths, and insert size can be varied according to individual preferences. No exact quantification of the cDNA is necessary prior to fragmentation, as both the amplification process and fragmentation reaction are extremely robust. Insert DNA is end-polished and subjected to a highly efficient ligation reaction with the included barcoded adapters, which can be purified by standard methods prior to quantification and sequencing. Amplification of the library with PCR is not necessary, so the production of PCR duplicates is avoided and library diversity remains high.