50 ng input total RNA was utilized for RNA-Seq library preparation utilizing the TruSeq Stranded RNA Access Library Prep Kit (cat# RS-301-2001). The method was automated on the Biomek FXP Laboratory Automation Workstation (Beckman Coulter). cDNA libraries were quantified utilizing the Quanti-iT PicoGreen assay (Life Technologies—cat# P7589). Libraries were also quality checked by Agilent Bioanalyzer using the High Sensitivity DNA kit (cat# 5067-4626). cDNA libraries were sequenced on the Illumina NextSeq500 platform as 75 bp paired end reads. The STAR RNA sequencing alignment tool (Spliced Transcripts Alignment to a Reference) aligner [STAR_2.5.0a]) was utilized to align the data to the genome, Homo sapiens UCSC hg19 (RefSeq gene annotations). DeSeq2 was used to perform differential expression analysis (± twofold, adj p value < 0.05). We computed overlaps between our differentially expressed gene set and the annotated Hallmark gene sets in the Molecular Signature DataBase, MSigDB (top 100, FDR q value < 0.05). Gene Cluster 3.0 was used to perform hierarchical clustering. Java TreeView (Version 1.1.5) software was used to plot the heatmap from hierarchical clustering results. The material presented here is original research, mRNA data has been uploaded at GEO (GEO: GSE136401. Token for reviewers: gzazgmomxdglnwb).
Biomek fxp laboratory automation workstation
Manufactured by Beckman Coulter
Sourced in United States
The Biomek FXP Laboratory Automation Workstation is a versatile liquid handling system designed to automate a wide range of laboratory workflows. It features modular hardware and software components, allowing for customization to meet specific laboratory needs. The Biomek FXP is capable of performing various liquid handling tasks, such as pipetting, aliquoting, and serial dilutions, with precision and accuracy.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate buffered saline (pbs), by Corning (3 mentions)
Proteinase k, by New England Biolabs (3 mentions)
1m dtt, by Merck Group (3 mentions)
Envision plate reader, by PerkinElmer (3 mentions)
Proteinase k, by Merck Group (2 mentions)
Fragment analyzer automated ce system, by Agilent Technologies (2 mentions)
Universal plus mrna seq kit, by Tecan (2 mentions)
Qubit dsdna hs kit, by Thermo Fisher Scientific (2 mentions)
Eb buffer, by Qiagen (2 mentions)
High sensitivity dna kit, by Agilent Technologies (1 mentions)
Nextseq 500 platform, by Illumina (1 mentions)
Quanti it picogreen assay, by Thermo Fisher Scientific (1 mentions)
Dapi 4 6 diamidino 2 phenylindole, by Merck Group (1 mentions)
Agencourt rnadvance tissue kit, by Beckman Coulter (1 mentions)
Bead ruptor elite, by Omni International (1 mentions)
Celltiter glo ctg luminescent cell viability assay, by Promega (1 mentions)
Poly 1 c lmw, by InvivoGen (1 mentions)
Nano glo hibit lytic detection system, by Promega (1 mentions)
Multidrop combi reagent dispenser, by Thermo Fisher Scientific (1 mentions)
Genetitan, by Thermo Fisher Scientific (1 mentions)
17 protocols using biomek fxp laboratory automation workstation
1
Automated RNA-Seq Library Preparation and Analysis
2
Efficient Genomic DNA Extraction from Cell Lines
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HEK293T cells were washed with 1X PBS (Corning) and lysed overnight by shaking at 55°C with 43.5 μL of gDNA lysis buffer (100 mM Tris-HCl at pH 8, 200 mM NaCl, 5 mM EDTA, 0.05% SDS) supplemented with 5.25 μL of 20 mg/ml Proteinase K (NEB) and 1.25 μL of 1M DTT (Sigma) per well for experiments in 96-well plates, or with 174 μL DNA lysis buffer, 21 μL Proteinase K, and 5 μL 1M DTT per well for experiments in 24-well plates. K562 cells were centrifuged for 5 min, media removed, and lysed overnight by shaking at 55°C with 174 μL DNA lysis buffer, 21 μL Proteinase K, and 5 μL 1M DTT per well in 24-well plates. U2OS cells and HeLa cells were washed with 1X PBS and lysed overnight shaking at 55°C with 174 μL DNA lysis buffer, 21 μL Proteinase K, and 5 μL 1M DTT per well in 24-well plates. Subsequently, gDNA was extracted from lysates using 1–2X paramagnetic beads as previously described5 (link) and eluted in 45 μL of 0.1X EB buffer. DNA extraction was performed using a Biomek FXP Laboratory Automation Workstation (Beckman Coulter).
3
Efficient gDNA Extraction from Cultured Cells
4
High-throughput PXR Activation Assay
5
RNA Extraction and Sequencing from Skeletal Muscle
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Skeletal muscle samples from the five PD subjects pre- and post-RT, along with 7 basal PD, 12 basal OA, and 12 basal YA (total n = 41, mean ± SD 7.5 ± 2.1 mg) were homogenized in a Bead Ruptor Elite bead mill homogenizer (Omni International, Kennesaw, GA, United States) at a speed of 4.2 m/s for 2 × 20 s while cooled by liquid nitrogen to 10°C. Muscle homogenates were processed using the Agencourt RNAdvance Tissue Kit (Beckman Coulter, Indianapolis, IN, United States) on a BioMek FXP Laboratory Automation Workstation (Beckman Coulter, Indianapolis, IN, United States). The quality (260/280: 1.9 ± 0.0, 260/230: 1.4 ± 0.1) and integrity (RIN: 8.95 ± 0.05, 28S/18S: 1.6 ± 0.2) of isolated RNA (125 ± 44 ng/μL) were assessed using NanoDrop and an RNA Standard Sensitivity Kit (DNF-471, Advanced Analytical Technologies, Ankeny, IA, United States) on a Fragment Analyzer Automated CE system (Advanced Analytical Technologies). Subsequently, cDNA libraries were constructed from 250 ng of total RNA using the Universal Plus mRNA-Seq kit (NuGEN Technologies, San Carlos, CA, United States). Library concentration (75.8 ± 17.9 ng/μL) was assessed fluorometrically using the Qubit dsDNA HS Kit (Thermo Fisher), and quality (average fragment size: 336 ± 5 bp) was assessed with the Genomic DNA 50Kb Analysis Kit (DNF-467, Advanced Analytical Technologies).
6
High-throughput Screening of Cancer Cell Lines
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The primary screen was performed in 384-well format using the CellTiter Glo (CTG) luminescent cell viability assay (Promega) as previously described27 . 3630 compounds from the St. Jude proprietary molecular glue library were screened against 9 human cancer cell lines (HD-MB03, MB004, MB002, MHH-CALL4, MOLM-13, TF-1, HEL, OCI-AML3, AML193). Each cell line was cultured in the complete medium recommended by the vendor and seeded in Corning 8804 BC white 384-well assay plates at densities of 1000, 1000, 1500, 7500, 1250, 156, 625, 1250, 1250 cells per well for HD-MB03, MB004, MB002, MHH-CALL4, MOLM-13, TF-1, HEL, OCI-AML, AML193, respectively. After overnight incubation at 37 °C in a humidified 5% CO2 incubator, cells were treated with compounds in dose–response format using a Pintool on a Biomek FXP Laboratory Automation Workstation (Beckman Coulter). After 72 h of incubation, cell proliferation was assessed using a CellTiter-Glo (CTG) luminescent cell viability assay (Promega) according to the manufacturer’s instructions. Luminescence signal was measured using an EnVision plate reader (PerkinElmer).
7
High-throughput siRNA screen for IFN and virus
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A549 wildtype or A549-IFN-Luc cells were transfected with siRNAs in 384-well format as previously described for A549 wildtype cells [11 (link)]. After 24 h, the A549-IFN-Luc cells were subjected to IFN induction assay as described below. After 48 h, the A549 wildtype cells were either subjected to cell viability assays (WST-1 assay, cell number determination assay) or virus replication assay as described below. All multi-well pipetting steps were performed using a Biomek FXP Laboratory Automation Workstation (Beckman Coulter). The siRNA library was composed of a validated human kinome library and a custom designed siRNA library (Qiagen) in total containing 3,482 siRNAs targeting 1,208 human genes. On each plate, control RNAs were included: non-targeting siRNA Allstars (14 wells) as negative control for all assays, polyinosinic-polycytidylic acid (poly I:C, final concentration: 2 μg/ml, 6 wells, poly(I:C)-LMW, Invivogen) as positive control for the IFN induction assay, an siRNA targeting the cellular gene PLK1 (siPLK1, 4 wells, [11 (link)]) as positive control for cell viability assays, and an siRNA targeting the IAV NP gene in case of IAV (8 wells, [77 (link)]) or an siRNA targeting the cellular gene XPO1 (8 wells, target sequence: 5’-CTGTGTCAGTTTGTAATGGAA-3’) in case of IBV, respectively, as positive control for virus replication assays. All siRNAs were purchased from Qiagen.
8
GSPT1 Protein Quantification in HEK293 Cells
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HEK293 hGSPT1 HiBiT tag cells were
seeded in white 384-well assay plates at a density of 800 cells per
well in triplicates. After overnight incubation, cells were treated
with compounds in a dose-response format using a Pintool on a Biomek
FXP Laboratory Automation Workstation (Beckman Coulter). After 4 h
of incubation, the level of GSPT1 HiBiT tag protein was evaluated
using the Nano-Glo HiBiT lytic detection system (Promega) according
to the manufacturer’s instruction. The luminescence signal
was measured using an EnVision plate reader (PerkinElmer).
seeded in white 384-well assay plates at a density of 800 cells per
well in triplicates. After overnight incubation, cells were treated
with compounds in a dose-response format using a Pintool on a Biomek
FXP Laboratory Automation Workstation (Beckman Coulter). After 4 h
of incubation, the level of GSPT1 HiBiT tag protein was evaluated
using the Nano-Glo HiBiT lytic detection system (Promega) according
to the manufacturer’s instruction. The luminescence signal
was measured using an EnVision plate reader (PerkinElmer).
9
Quantifying Antibody-Drug Conjugate Stability
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Following labeling, 10 μL was
transferred into a 384-well plate and diluted with 90 μL of
PBS or plasma containing 1.8 mg/mL ethylenediaminetetraacetic acid
and 10 mM sodium azide. The different plasmas (BioreclamationIVT,
Baltimore, MD) used were from CD1 mice, Sprague Dawley rats, cynomolgus
monkeys, and humans.
The ADC-containing buffer/plasma samples
were incubated at 37 °C with 5% CO2. Samples were
collected after incubation times of 0, 4, 24, 48, 72, and 144 h with
a Biomek FXP Laboratory Automation Workstation (Beckman Coulter, Indianapolis,
IN). The plasma plates were retrieved from the Cytomat Plate Hotel
in the incubator. Five microliters from each well was transferred
to two 384-well plates, with one containing 70 μL of 95:5 acetonitrile/methanol,
with 50 nM carbutamide as the internal standard. Using a Multi-drop
Combi Reagent Dispenser (Thermo Scientific), 20 μL of water
and 5 μL of DMSO were added to the plate containing the organic
solvent. Plates were spun at 3000 rpm for 5 min at 4 °C prior
to further analysis.
transferred into a 384-well plate and diluted with 90 μL of
PBS or plasma containing 1.8 mg/mL ethylenediaminetetraacetic acid
and 10 mM sodium azide. The different plasmas (BioreclamationIVT,
Baltimore, MD) used were from CD1 mice, Sprague Dawley rats, cynomolgus
monkeys, and humans.
The ADC-containing buffer/plasma samples
were incubated at 37 °C with 5% CO2. Samples were
collected after incubation times of 0, 4, 24, 48, 72, and 144 h with
a Biomek FXP Laboratory Automation Workstation (Beckman Coulter, Indianapolis,
IN). The plasma plates were retrieved from the Cytomat Plate Hotel
in the incubator. Five microliters from each well was transferred
to two 384-well plates, with one containing 70 μL of 95:5 acetonitrile/methanol,
with 50 nM carbutamide as the internal standard. Using a Multi-drop
Combi Reagent Dispenser (Thermo Scientific), 20 μL of water
and 5 μL of DMSO were added to the plate containing the organic
solvent. Plates were spun at 3000 rpm for 5 min at 4 °C prior
to further analysis.
10
Transcriptome Analysis via Affymetrix Microarray
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Messenger RNA analysis was performed on purified RNA converted to biotin-labeled complementary RNA copies with the Affymetrix HT 3’ IVT Plus kit (Affymetrix, Santa Clara, CA) per protocol provided at a Beckman Biomek FXP Laboratory Automation Workstation (Beckman, Indianapolis, IN). Briefly, 250 ng of total RNA were reverse-transcribed into complementary DNA copies using oligo-dT primers and reverse transcriptase followed by second-strand synthesis using DNA polymerase I [2 (link)]. After purification, the cDNA library was used as a template to generate biotin-labeled cRNA copies using T7 RNA polymerase and biotinylated deoxyuridine triphosphate. Biotinylated cRNA was fragmented by limited alkaline hydrolysis and then hybridized overnight to Affymetrix GeneTitan U219 array plates using the Affymetrix GeneTitan instrument and protocol. After processing, chip images were converted to numeric data with the probe logarithmic intensity error algorithm as executed in the Affymetrix GeneChip Expression Console. All data were MIAME compliant.
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