The samples were thawed and centrifuged at 15,000× g at 4°C for 10 min. The supernatant was mixed with 2% methanol at a 1:1 ratio and filtered through a 0.45 μm filter (Cat No. HAWP04700; Millipore, Billerica, MA) before analysis. The cordycepin concentration in the supernatant was measured using high performance liquid chromatography with a UV detector at 260 nm (Shimadzu, Tokyo, Japan). The TSK‐gel ODS‐80Ts (Tosoh Corp., Tokyo, Japan) was used at 40°C with 0.1% phosphoric acid: methanol at a 98:2 ratio (v/v) as the mobile phase (Masuda, Das, Hatashita, Fujihara, & Sakurai, 2014 ). cordycepin (Wako Pure Chem. Ind. Ltd., Osaka, Japan) was used as the reference standard.
Hawp04700
Manufactured by Merck Group
Sourced in United States, Australia, Germany
The HAWP04700 is a laboratory equipment product manufactured by Merck Group. It serves as a core function for laboratory applications, but a detailed unbiased description cannot be provided while maintaining the requested parameters.
Automatically generated - may contain errors
Lab products found in correlation
Cordycepin, by Shimadzu (2 mentions)
Tsk gel ods 80ts, by Tosoh (2 mentions)
Dissection microscope, by Leica (1 mentions)
Ap1004700, by Merck Group (1 mentions)
Puromycin, by Thermo Fisher Scientific (1 mentions)
Transit lt1, by Mirus Bio (1 mentions)
Lba sm km plates, by Corning (1 mentions)
Gttp04700, by Merck Group (1 mentions)
Gibco opti mem, by Thermo Fisher Scientific (1 mentions)
X tremegene hp dna transfection reagent, by Merck Group (1 mentions)
Lenticrispr v2 vector, by Addgene (1 mentions)
C18 column, by Phenomenex (1 mentions)
Filter flask, by Merck Group (1 mentions)
5 ml bead beating tube, by Qiagen (1 mentions)
Magnetic filter funnel, by Pall Corporation (1 mentions)
Zr bashingbead lysis tube, by Zymo Research (1 mentions)
Qiaamp viral rna mini kit, by Qiagen (1 mentions)
Dna rna shield, by Zymo Research (1 mentions)
Histrap ff crude column, by Cytiva (1 mentions)
Kta pure chromatography system, by Cytiva (1 mentions)
22 protocols using hawp04700
1
Cordycepin Quantification in Supernatant
2
Wastewater SARS-CoV-2 Detection Protocol
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Bovine Coronavirus (BCoV, ValleyVet Supply, Marysville, KS) were spiked at a concentration of 6300 copies per mL of wastewater sample prior to concentration as overall process control. Wastewater samples were adjusted to a pH of 3.5–4 using 10 M HCl, followed by the addition of 2.5 M MgCl2.6H2O to achieve a final concentration of 25 mM MgCl2.6H2O (Ahmed et al., 2020b (link); Cashdollar and Wymer, 2013 (link)). Using a disposable filter funnel fitted with a 47 mm dia. 0.45 μm type HA Filter (47 mm diameter, 0.45 μm type HA, HAWP04700 Millipore, Bedford MA), 20 mL of each wastewater sample was concentrated using a vacuum filtration manifold and was filtered to dryness. Negative process control or Method Blank consisting of 1× phosphate buffered saline (PBS) was filtered during each of the sample processing events using a new sterile filter funnel and type HA electronegative filter (Ciesielski et al., 2021 (link)). After wastewater concentration, the filter was placed in individual 2 mL microcentrifuge tubes. The process was repeated 8 times for each wastewater sample. One filter was used for Workflow 1, one for Workflow 2, (Fig. 2 ) and the others were archived at −80 °C for future analyses. For workflow 1 the filter was suspended in the AVL buffer for RNA extraction.Fig. 2 ![]()
Showing the two different workflows performed to quantify SARS-CoV-2 in the influent wastewater.
3
Nitrocellulose Filter Cell Culturing
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In these assays, 1 × 107 cells were plated and allowed to develop on 47mm nitrocellulose filters (Millipore, HAWP04700) over 24 h [74 ]. Each filter was placed on an absorbent 3M paper pad (Millipore, AP1004700) soaked in phosphate buffer of varying pH. Phosphate buffer was prepared using varying amounts of KH2PO4 and K2HPO4 to modulate pH without altering ionic strength. Subsequent developmental phenotypes were imaged using a dissection microscope (Leica) and a QICAM FAST 1394 camera (QImaging).
4
Lentiviral Transduction for Stable Cell Lines
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To prepare lentivirus used for the generation of stable cell lines, HEK293T cells were transiently transfected with lentiviral packaging vectors and pHAGE-mCherry-RTN3L using TransIT-LT1 (Mirus; MIR2306; Madison, WI). The cells were harvested 2 days post-transfection and the supernatant was passed through a 0.45 µm syringe filter unit (Millipore; HAWP04700; Burlington, MA) and frozen at –80°C. Subsequently, control and LNPK KO U2OS cells were transduced with lentivirus and selected with 1.5–2 µg/ml of puromycin (Gibco; A1113803).
5
Construction of transposon mutant library
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The Mariner-based Himar1 Tn (which inserts into TA sites) and a previously published protocol [26 (link),30 (link)] were used to construct two independent libraries for each condition assayed. Few changes were made in order to optimize the saturation of our transposon mutant library. An initial Tn-library was created in LBA by mixing washed bacteria from 1 ml of an overnight culture of Tn donor and receptor (Table S1) in a final volume of 500 µl of fresh LB at a ratio of 1:1. Then, 50 µl-aliquots were spotted onto 0.45 µm-filters (Millipore, HAWP04700) placed on LBA plates that were incubated for 6 h. After conjugation, the filters were recovered and grown bacteria were spread onto LBA+Sm+Km plates of 24.5 × 24.5cm [2 (link)] (Corning) that were incubated at 37ºC for 24 h.
Bacteria grown on the plates (approx. 1.5 million) were scrapped with 12 ml of fresh LB and 3 ml were used to obtain genomic DNA exactly as Chao et al. described [26 (link),27 (link)] and the rest (around 20 ml) was kept with 20% glycerol at -80 ºC. DNA from this library was used for determination of genes required for V. vulnificus growth in vitro
Bacteria grown on the plates (approx. 1.5 million) were scrapped with 12 ml of fresh LB and 3 ml were used to obtain genomic DNA exactly as Chao et al. described [26 (link),27 (link)] and the rest (around 20 ml) was kept with 20% glycerol at -80 ºC. DNA from this library was used for determination of genes required for V. vulnificus growth in vitro
6
Gut and Water Sampling for Larvae
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For each of the aquaria/chambers, the gut samples from four larvae were collected on days 14, 21 and 28 (Table S2 , sample meta-data), and the water was also sampled from each aquarium/chamber after filtering 1 L of water through 0.45- and 0.2-μm membrane filters (Millipore HAWP-04700 and Millipore GTTP-04700). All samples were frozen in liquid nitrogen and stored at −80 °C until further analysis. All gut and water samples were stored and individually analysed. The detailed protocols on gut and water sampling are described in Giatsis et al.43 (link).
7
Quantification of Cordycepin in Samples
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Samples were thawed and centrifuged at 15,000×g at 4°C for 10 min. The supernatant was mixed with 2% methanol at a 1:1 ratio and filtered through a 0.45 μm filter (Cat No. HAWP04700, Millipore, Billerica, MA, USA) before analysis. The cordycepin concentration in the supernatant was measured by high-performance liquid chromatography (HPLC) with an UV detector at 260 nm (Shimadzu, Tokyo, Japan). The TSK-gel ODS-80Ts (Tosoh Corp., Tokyo, Japan) was used at 40°C with 0.1% phosphoric acid: methanol at a 98:2 ratio (v/v) was used as the mobile phase [8 ]. cordycepin (Wako Pure Chem. Ind. Ltd., Osaka, Japan) was used as the reference standard.
8
Wastewater Viral Concentration Protocol
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Prior to wastewater concentration, the pH of the wastewater samples was adjusted to 3.5 with 10 M HCl solution followed by inoculation with MgCl2 x 6 H2O (to a final concentration of 25 mM). Magnetic stir bars were added to the samples and a magnetic stir plate was used to agitate until thoroughly mixed. 50 mL of sample was then filtered to dryness through a 47 mm dia., 0.45 μm Mixed Cellulose Ester (HA, HAWP04700, Millipore Corp., Bedford, MA) filter using vacuum filtration manifolds. Using sterilized forceps, HA filters were transferred to a microcentrifuge tube and immediately analyzed or stored at −80 °C for no more than 1 week.
9
Generation of Epigenome and Kinome CRISPR Libraries
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The epigenome and kinome sgRNA libraries were a gift from Dr. B. Evers and were previously described in Evers et al. [22 (link)]. In short, the epigenome sgRNA library consisted of 5130 sgRNAs targeting a total of 446 genes encoding epigenetic regulators, and the kinome sgRNA library consisted of 5860 sgRNAs targeting 504 genes encoding human kinases, with both libraries containing ≥10 sgRNAs per gene [22 (link),23 (link)]. The sgRNA plasmid library was generated by cloning all sgRNAs into lentiCRISPR v2 vectors (Addgene #52961, Watertown, MA, USA) containing an U6 promoter A complete list of all gene-specific sgRNAs, as well as positive and negative (non-targeting) controls is provided in Supplementary Table S1 . Virus was generated by transfection of HEK293T cells using library plasmid, MD2.G plasmid (Addgene #12259), PAX2 plasmid (Addgene #12260), and X-tremeGENE™ HP DNA Transfection Reagent (Sigma-Aldrich #XTGHP0RO, St. Louis, MO, USA). The medium was replaced by Gibco Opti-MEM (ThermoFisher #31985070, Waltham, MA, USA) the following day. Virus was harvested two days after transfection, filtered through a 0.45 µM low protein binding membrane (Millipore #HAWP04700, Burlington, MA, USA), and concentrated using vivaspin-20 columns (Sigma-Aldrich #Z614653). Concentrated virus was stored in aliquots at −80 °C for further use.
10
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