Blood samples were collected from 300 Cambodian individuals, who came from 16 of the 22 regions of Cambodia (Fig. 1 ), but lived in Thailand at the time of the sample collection. The study was approved by the Thai Ministry of Public Health in accordance with international ethical standards. According to the Declaration of Helsinki, participation in the study was on a voluntary basis and informed consent was obtained from all donors (Chiang Mai University, Thailand, January 11, 2007). Detailed information on the geographical origin and ethnic background of the samples including the maternal region of birth was collected from all samples and can be found in Table 1 and Supplemental Table S1 . Genomic DNA was extracted on a BioRobot EZ1 advanced Workstation (QIAGEN, Hilden, Germany) and quantified on an Infinite M200 NanoQuant (Tecan Group Ltd., Männedorf, Switzerland). On each plate 94 DNA samples were normalized in their DNA-concentration and arrayed on a 96-well plate with a TECAN Freedom EVO platform (Tecan Group, Männedorf, Switzerland), leaving space for two no-template controls per plate. Thereby, three full 96-well plates and one 96-well plate with the remaining DNA wells were generated. See Supplemental Material Fig. 1 with a graphical representation of the workflow.
Freedom evo platform
Manufactured by Tecan
Sourced in Switzerland
The Freedom EVO platform is a modular, flexible, and automated liquid handling workstation. It is designed for a wide range of laboratory applications, including sample preparation, assay setup, and reagent dispensing. The platform features precise liquid handling capabilities and can be customized with a variety of modules and accessories to meet the specific needs of the laboratory.
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Lab products found in correlation
9 protocols using freedom evo platform
1
Cambodian DNA Samples Collection
2
Automated Mitochondrial Genome Amplification
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The entire mitochondrial genome was amplified in two overlapping fragments of approximately 9 kb each26 (link). For amplifying the mtDNA of one 96-well DNA plate, two 96-well plates containing PCR-mastermixes were prepared: one plate for fragment A and one plate for fragment B. Then, the DNA was transferred from the DNA-plate to the PCR-plate with a TECAN Freedom EVO platform (Tecan Group, Männedorf, Switzerland).
The DNA was amplified in a total reaction volume of 50 µl, containing approximately 280 ng (fragment A) or 120 ng (fragment B) of DNA, 1 µl of Herculase II Fusion DNA Polymerase (Stratagene, La Jolla, USA), 10 µl of 5xPCR reaction buffer, 250 µM each dNTP (Stratagene, La Jolla, USA), and 0.25 µM each primer. The PCR reaction was done in a 96-well thermal cycler (Bio-Rad Laboratories GmbH, Munich, Germany). A incubation at 95 °C for two minutes was used for initial denaturation. For a high specificity 10 touch-down cycles were applied with 95 °C for 20 s, 69 °C–0.5C°/cycle for 20 s, and 72 °C for 4 min 30 s. Subsequently, amplification was performed using 40 PCR cycles (95 °C 20 s, 64 °C 20 s and 72 °C 10 min)26 (link). PCR products were purified with the QiaVac (QIAquick Multiwell PCR Purification Kit; Qiagen, Venlo, Netherlands). The post-PCR pipetting steps were performed with a Plate Mate 2 × 2 Automated Liquid Pipettor (Matrix Technologies Corp., Hudson, USA).
The DNA was amplified in a total reaction volume of 50 µl, containing approximately 280 ng (fragment A) or 120 ng (fragment B) of DNA, 1 µl of Herculase II Fusion DNA Polymerase (Stratagene, La Jolla, USA), 10 µl of 5xPCR reaction buffer, 250 µM each dNTP (Stratagene, La Jolla, USA), and 0.25 µM each primer. The PCR reaction was done in a 96-well thermal cycler (Bio-Rad Laboratories GmbH, Munich, Germany). A incubation at 95 °C for two minutes was used for initial denaturation. For a high specificity 10 touch-down cycles were applied with 95 °C for 20 s, 69 °C–0.5C°/cycle for 20 s, and 72 °C for 4 min 30 s. Subsequently, amplification was performed using 40 PCR cycles (95 °C 20 s, 64 °C 20 s and 72 °C 10 min)26 (link). PCR products were purified with the QiaVac (QIAquick Multiwell PCR Purification Kit; Qiagen, Venlo, Netherlands). The post-PCR pipetting steps were performed with a Plate Mate 2 × 2 Automated Liquid Pipettor (Matrix Technologies Corp., Hudson, USA).
3
Turbidity Measurements of Proteins
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Turbidity measurements were set up using a liquid handler (Freedom EVO platform, Tecan) in flat bottom UV-STAR 384 well plates (#781801, Greiner BioOne, Frickenhausen, Germany). For assays, reaction buffer, storage buffer for adjustment, and artificial crowding agent were added to individual wells and mixed before the addition of the proteins after which the mixture was mixed again by pipetting. All reactions were set up in triplicates on the plate. Protein was thawed immediately before addition and the protein and the 384-well plate were cooled to 4 °C during the liquid handling process. All experiments were performed at a constant final buffer composition of 50 mM HEPES pH 7.25, 50 mM KCl, 3% Glycerol, 7 mM MgCl2 with or without artificial crowder (final concentration given in % w/v). Subsequently, the turbidity was determined via absorption at 340 nm using a plate reader (model Spark, Tecan).
4
Automated Fungal Metabolome Extraction
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Samples for fungal metabolome analysis were prepared on an automated platform that combined both extraction and partitioning steps. Fungal cultures prepared in 16- by 100-mm borosilicate tubes were placed on a Tecan Freedom EVO platform and 3 ml of ethyl acetate was added to each sample. After extraction for 4 h, 3 ml of water was added to each tube to facilitate the partitioning process. Aliquots consisting of 2 ml of the upper ethyl acetate layers were transferred to deep-well 96-well plates. While the ethyl acetate was being removed from the samples in vacuo, the fungal culture tubes were each charged with an additional 3 ml of ethyl acetate to continue the partitioning process. The plates were returned to the liquid handler platform, at which point a second set of 2-ml aliquots of ethyl acetate was removed from the tubes and deposited into the deep-well 96-well plates. The organic solvent was removed in vacuo and the remaining organic residues were stored at −20°C for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.
5
High-throughput Screening for Pyrin-specific Modulators
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All robotic steps were performed on a Tecan Freedom EVO platform. Compounds from the Prestwick Chemical Library® were evaluated at a 1:1,000 or 1:2,000 dilution of the original stock for plates 1–14 and plate 15, respectively (see Table S1 ). 1 μL of DMSO solutions was spiked into dry well of F-bottom clear cell culture treated 96-wells plates (Greiner Bio One), with columns 1 and 12 devoted to controls and used to calculate the Z′-factor. U937 cells expressing p.S242R MEFV were treated or not (counterscreen) with doxycycline (1 μg/mL) for 16 h, centrifuged and seeded at 105 cells per well (100μL final volume) in RPMI 1640 without phenol red, 10% FCS, 1mM HEPES, 1% PSA, 1mM Glutamine, in the presence of propidium iodide at 5 μg/mL. Following 90 min of incubation at 37°C, fluorescence intensity (excitation wavelength at 535 nm and emission wavelength at 635 nm) corresponding to propidium iodide incorporation was measured on a microplate reader (Infinite M1000, Tecan). The average Z′ value was 0.67 ± 0.12, indicating a robust and reliable assay. Mean fluorescence + 3SD was retained as a threshold. Compounds triggering cell death only in the presence of doxycycline were considered as pyrin-specific and defined as hits.
6
High-Throughput Screening of Anti-TB Compounds
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A high-throughput whole-cell screening of a chemical library composed of 35 860 compounds was performed on a Freedom EVO platform (Tecan), using M. aurum ATCC23366 as a surrogate of M. tuberculosis (38 (link)). A nNF compound from the Institut Curie chemical library (part of the ‘Chimiothèque Nationale’ (39 (link))) was identified as a potent anti-tuberculosis hit (nNF-C18; Figure 1 and Supplementary Table S1 ) and nineteen additional derivatives belonging to the same chemical library were selected (nNF-C1 to nNF-C20; Figure 1 and Supplementary Table S1 ). In addition, 13 desnitro analogues were also chosen from the same library (derivatives C1B to C13B; Supplementary Table S2 ). nNF-C1- nNF-C20 and C1B-C13B comprised the chemical set of compounds for analysis in our study. The chemical synthesis of all chemical derivatives was previously described (40–48 ). Compounds were dissolved in DMSO, protected from light, and stored at –20°C until use.
7
High-throughput Screening for Pyrin-specific Modulators
8
Genetic Determinants of Type 2 Diabetes
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Peripheral venous blood samples were collected from each participant. Genomic DNA was extracted from peripheral blood leukocytes on a Tecan Freedom EVO platform (Tecan, Switzerland) using the Mag-Bind Blood DNA Kit (CWBIO, China). Single nucleotide polymorphisms (SNPs) genotyping work was performed using a custom-by-design 48-Plex SNPscanTM Kit (Cat#:G0104; Genesky Biotechnologies Inc., Shanghai, China). SNPs were selected from their consistent associations with impaired insulin synthesis, secretion, action, or other T2D-related traits in genome-wide association studies and candidate gene studies, especially in Chinese or Asian, and had to meet the following criteria: fitting Hardy-Weinberg equilibrium, genotyping success rates exceed 95%, and the genotyping concordance of replicated quality-control samples exceed 95%. On these basics, 25 SNP were incorporated in our study [27 (link)–41 (link)]. The details of these SNPs are shown in Supplementary Table 2 .
9
ASCUS/CIN1 Cervical Cytology Validation
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One hundred consecutive collected cervical cytology samples with a diagnosis of ASCUS or CIN1 were included in the study. Samples were collected from the Department of Laboratory Medicine at Örebro University Hospital in 2012. ThinPrep vials holding 20 mL, from either primary screening samples (n = 44) or follow-up samples after a previous diagnosis (n = 56), were used. All included women were aged 35 years or older and their samples were forwarded for HPV testing according to the national recommendations. Upon inclusion in the validation study, all samples were deidentified and given a serial number that could not be connected to either personal data or clinical evaluation. No informed consent was obtained since this was a purely methodological study using anonymized patient samples without any connection to patient data or patient identity. DNA and mRNA analysis was performed on all cases in parallel before and after biobanking.
Samples were transferred from ThinPrep vials to 96-well plates using the Freedom EVO platform (Tecan) according to Perskvist et al (11) . In short, primary sedimentation of samples was performed and 4 mL was pipetted to an intermediate tube. After a second sedimentation step, a cell volume of 500 µL was transferred to the repository plate. Plates were kept in a -20° freezer for 2 to 6 months and thawed before secondary DNA or mRNA analysis.
Samples were transferred from ThinPrep vials to 96-well plates using the Freedom EVO platform (Tecan) according to Perskvist et al (11) . In short, primary sedimentation of samples was performed and 4 mL was pipetted to an intermediate tube. After a second sedimentation step, a cell volume of 500 µL was transferred to the repository plate. Plates were kept in a -20° freezer for 2 to 6 months and thawed before secondary DNA or mRNA analysis.
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