Benchmarking RNA-seq Protocols with ERCC Spike-ins

We used six datasets representing the TruSeq, Smart-Seq and UMI-seq protocols and varying amounts of starting material from bulk RNA or single cell RNA. All analysed datasets contain the ERCCs spike-in RNAs. This is a set of 92 artifical poly-adenylated RNAs designed to match the characteristics of naturally occurring RNAs with respect to their length (273–2022 bp), their GC-content (31–53%) and concentrations of the ERCCs (0.01–30,000 attomol/μl). The recommended ERCC spike-in amounts result in 5–10⁷ ERCC RNA molecules in the cDNA synthesis reaction.
To reduce biological variation, we used the well-characterized Universal Human Reference RNA (UHRR; Agilent Technologies) for the two datasets produced for this study. We downloaded UHRR- and HBRR-TruSeq data from SEQC/MAQC-III2 (link). Finally, we also analyse the single cell data published in Wu et al.20 (link), for which the colorectal cancer cell-line HCT116 was used (Table 1). The input mostly being commercially distributed human samples, we expect all biological samples analysed in this study to have similarly high quality and complexity. All data that were generated for this project were submitted to GEO under accession GSE75823.