All materials, reagents and equipment used in both the manual and automated workflows are summarized in Table S1.
These are referred to as Biomek-1 and Biomek-2 in the Results and Discussion section. Sequencing of the libraries prepared by automation was carried out twice to assess run-to-run sequencing reproducibility and variance. The library pool was subsequently clustered with the HiSeq 3000/4000 SR Cluster Kit on a cBot and sequenced on a HiSeq 4000 Sequencing System (Illumina) with dual index, single read at 85 bp length (Read parameters: Rd1: 86, Rd2: 8, Rd3: 8), reaching an average depth of 39.7 million Pass-Filter reads per sample (9.2% CV). All sequencing libraries were then normalized on the MicroLab STAR (Hamilton), pooled, and spiked in with PhiX Control v3 (Illumina). Libraries were also assessed for size distribution and adapter dimer presence by the High Sensitivity Small Fragment DNF-477 Kit on a 96-channel Fragment Analyzer (Agilent). The manual handling also included several clean-up or mixing steps that were performed using both the benchtop VIAFLO 384 pipetting system with 12.5 μL or 125 μL pipetting heads (Integra) and EpMotion using 50 μL and 300 μL tips (Eppendorf).įinal sequencing libraries were quantified by the High Sensitivity dsDNA Quanti-iT Assay Kit (ThermoFisher) on a Synergy HTX (BioTek). The various incubation and PCR amplification steps were performed on an Eppendorf X50 MasterCycler for the manual workflow, and on an integrated on-deck automated thermal cycler (ATC) for the Biomek workflow (Fisher Scientific). Both methods followed the manufacturer's protocol, except for the use of AMPure XP beads (Beckman Coulter) in the clean-up steps, instead of the recommended SPRIselect Beads or NEBNext Sample purification beads. The manual workflow lasted 12 hours and spanned across two days with 7.5 hours hands-on time, while the automated overnight method required an initial 2 hours deck loading time and a subsequent 9 hours hands-free run time ( Fig. 1). Libraries were either processed manually or on a Biomek i7 Hybrid instrument (Beckman Coulter) using a final 12 and 13 index PCR cycles, respectively. A total RNA input of 250 ng was used for library construction with the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina, together with the NEBNext Poly(A) mRNA Magnetic Isolation Module upstream and the NEBNext Multiplex Oligos for Illumina downstream (all New England Biolabs).
Total RNA samples (n=84) were normalized on the MicroLab STAR automated liquid platform (Hamilton).
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