Zephyr g3 spe workstation

Cells in plates were incubated at 72°C for 3 min. A reverse transcription master mix was added to the samples containing 1 µM of LNA-oligonucleotide (5’-AGCAGTGGTATCAACGCAGAGTACATrGrG+G-3’; Exiqon), 6 µM MgCl₂, 1M Betaine (Affymetrix), 1X reverse transcription buffer, 50 µM DTT, 0.5 U of SuperRNAsin, and 0.5 µL of reverse transcriptase (Table 1). The total volume of the reaction was 10 µL. The plate was incubated using the following programme: 1 cycle of 42°C for 90 min; 10 cycles (42°C/2 min, 50°C/2 min); 1 cycle of 70°C for 15 min. Samples were then supplemented with 1X KAPA Hotstart HiFi Readymix and 2.5 µM of the ISO SMART primer (Picelli et al., 2013 (link)) and incubated using the following cycling programme 1 cycle of 98°C for 3 min; 25 or 30 cycles (98°C/20 s, 67°C/15 s, 72°C/6 min); 1 cycle of 72°C for 5 min (Table 1). Samples were then purified with 1X Agencourt Ampure beads (Beckman Coulter) in a Zephyr G3 SPE Workstation (Perkin Elmer) according to the manufacturer’s recommendation. Amplified cDNA was eluted in 10 µL nuclease-free water. Details of different permutations of the protocol tested during the optimisation process are given in Table 1.

Initial validation studies used supported liquid extraction (SLE) optimized for 48-wellplate processing on a PerkinElmer Zephyr G3 SPE Workstation (Waltham, MA). Sample extracts were analyzed using an Agilent 1260 quaternary liquid chromatography system (Santa Clara, CA) coupled to an Agilent 6420 tandem mass spectrometer (LC-MS/MS). Instrument control and data acquisition relied on MassHunter LC/MS Data Acquisition (VER B.08.00). Data analysis was performed using MassHunter Quantitative Analysis (VER B.07.01 SP2). Specific equipment used for inter-laboratory comparisons varied between different testing facilities (Supplemental Tables S1 – S5).

Supported liquid extraction (SLE) procedures were optimized for 96-wellplate processing on a PerkinElmer Zephyr G3 SPE Workstation (Waltham, MA, United States). Sample extracts were analyzed using an Agilent 1260 quaternary liquid chromatography system (Santa Clara, CA, United States) coupled to an Agilent 6420 tandem mass spectrometer (LC-MS/MS). Instrument control and data acquisition relied on MassHunter LC/MS Data Acquisition (VER B.08.00). Data analysis was performed using MassHunter Quantitative Analysis (VER B.07.01 SP2).