Small RNA-seq is increasingly being used for profiling of small RNAs. Quantitative characteristics of long RNA-seq have been extensively described, but small RNA-seq involves fundamentally different methods for library preparation, with distinct protocols and technical variations that have not been fully and systematically studied. We report here the results of a study using common references (synthetic RNA pools of defined composition, as well as plasma-derived RNA) to evaluate the accuracy, reproducibility and bias of small RNA-seq library preparation for five distinct protocols and across nine different laboratories. We observed protocol-specific and sequence-specific bias, which was ameliorated using adapters for ligation with randomized end-nucleotides, and computational correction factors. Despite this technical bias, relative quantification using small RNA-seq was remarkably accurate and reproducible, even across multiple laboratories using different methods. These results provide strong evidence for the feasibility of reproducible cross-laboratory small RNA-seq studies, even those involving analysis of data generated using different protocols. (Introduction without separate heading below)RNA-seq using next generation sequencing has been a transformative technology that has been widely used as a method for characterizing the transcriptome in a wide range of biological contexts 1,2 . Applications of RNA-seq fall into two categories: long RNA-seq and small RNA-seq, distinguished not only by the size of the targeted RNAs, but also by the technical methods used and the resulting biases of the different approaches in the quantitative data produced 3 . For example, the production of the libraries for long RNA-seq, by virtue of having sufficiently long target RNA lengths, commonly utilizes primers (e.g., random primers or oligo-dT) for direct generation of cDNA from RNA. However, small RNA-seq library construction methods typically require an RNA ligation or polyA tailing step to overcome the challenge of performing reverse transcription and subsequent PCR-based amplification from extremely short (e.g., 16-30 nt) target RNA sequences.Multiple approaches have been developed to overcome the challenge of uniformly and robustly generating cDNA from small RNAs for the purpose of small RNA-seq library preparation 4-9 . Protocols in use for small RNA-seq therefore vary more widely than those used for long RNA-seq, creating significantly more potential for variation across small RNA-seq results using different library preparation protocols and by different labs. In addition, small RNA-seq is increasingly used to study small RNAs present in very low input concentration samples (e.g., in exosomes and other types of extracellular vesicles (EV) 10-19 , or in RNA-protein complexes present in biofluids 20-26 ). Normalization methods 27-29 developed to correct for variation in long RNA-seq data are typically not well-suited for such small RNA-seq data, making it even more important to

Summary Several new complexes of platinum with positively charged cellular dyes have been synthesised in an effort to find chemotherapeutic drugs with increased antitumour cytotoxicity. As part of this effort, the direct cytotoxicities of some of these complexes as well as their ability to inhibit bleomycin potentially lethal damage repair (PLDR) was studied in vitro in a squamous cancer cell line of human origin . All of the new agents were more cytotoxic against exponentially growing than against plateau phase cell cultures. Exposure of cells to non-lethal drug concentrations for between 1 and 6h led to measurable inhibition of bleomycin PLDR in the case of each drug tested. In order of decreasing ability to inhibit bleomycin PLDR, Pt(fast black)2, Pt(thioflavin)2 and Pt(thionin)2 were more effective than CDDP, while Pt(methylene blue)2, Pt(Rh-123)2 and Pt(pyronin y)2 were less effective. The most directly cytotoxic agents were Pt(thioflavin)2, Pt(pyronin Y)2 and Pt(Rh-123)2 which also proved to be the least selectively toxic drugs towards exponential versus plateau phase cells. These results indicate that several of the new platinum complexes may be effective cytotoxic agents as well as effective inhibitors of DNA repair process following exposure of cells to other DNA interactive modalities.The glycopeptide antibiotic bleomycin has demonstrated clinical usefulness in the treatment of squamous cell cancer of the head and neck (SCCHN) (Turrisi et al., 1978), testicular tumours (Einhorn & Donahue, 1977) and lymphomas (Coltman et al., 1978). It is used in combinations with other agents because bleomycin does not exhibit dose limiting haematopoietic toxicity (Hubbard et al., 1975). When used as a single agent, however, complete response rates are relatively low and the emergence of drug resistance is a common problem (Crooke & Bradner, 1976).To various extents, mammalian tumour cells have the capacity to repair drug-and radiation-induced damage. The ability of cells to recover from potentially lethal damage has been modelled in vitro by maintaining cells in conditions which prevent them from proliferating for various times and thereby allowing time for repair processes to take place (Barranco & Townsend, 1986).Solid tumours and slow growing lymphomas are likely to contain large populations of non-cycling cells, which may have the capacity to repair potentially lethal damage and contribute to regrowth of the tumour. An in vitro system containing cells in stationary phase may be more analogous to the in vivo situation than are cells in exponential growth. Such an experimental model can be created by growing monolayer cell cultures to confluency under conditions of constant medium renewal without subculture. These stationary phase cultures contain a large fraction of non-cycling, but potentially clonogenic, cells (Hahn & Little, 1972). In such model systems, time dependent enhancement of cell survival observed with longer pre-subculture intervals following exposure to cytotoxic agents can be inferred as due to poten...