Lysis:Article Title: Quantitative mapping of pseudouridines in bacterial RNA
Article Snippet: .. The reaction was immediately stopped using stop solution (95% ethanol +5% Qiazol lysis solution (Qiagen, #79306)) followed by snap freezing in liquid nitrogen. .. The samples were allowed to thaw on ice, centrifuged, and bacterial pellets were resuspended in TE buffer containing 15 mg/ml lysozyme and 20 μl/ml proteinase K. To this mixture, 2 μl of a 1/10 of ERCC RNA spike-in sequences (Invitrogen, #4456740) was added for downstream normalization.
Article Title: Quantitative mapping of pseudouridines in bacterial RNA.
Article Snippet: .. The reaction was immediately stopped using stop solution (95% ethanol + 5% Qiazol lysis solution (Qiagen, #79306)) followed by snap freezing in liquid nitrogen. .. The samples were allowed to thaw on ice, centrifuged, and bacterial pellets were resuspended in TE buffer containing 15 mg/ml lysozyme and 20 ul/ml proteinase K. To this mixture, 2 ul of a 1/10 of ERCC RNA spike-in sequences (Invitrogen, #4456740) was added for downstream normalization.
SYBR Green Assay:Article Title: Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics
Article Snippet: .. STOP solution (Qiagen, cat. no. 1032393) Nuclease-free water (Fisher Scientific, cat. no. BP2484-100) Random hexamers, 50-μM (Integrated DNA Technologies (IDT); see Reagent Setup) DMSO (Sigma-Aldrich, cat. no. D8418-100ML) SYTO 13, 5 mM (Invitrogen, cat. no. S7575) DTT, 1 M (Sigma-Aldrich, cat. no. 646563-10X.5ML; see Reagent Setup) Household bleach (3–8% (wt/vol) solution of sodium hypochlorite) Phylogenetic screening Ultrapure water, such as Milli-Q water (Millipore) or other filtered and deionized water (DIW) SsoAdvanced SYBR Green Supermix (Bio-Rad, cat. no. 172-5264) 926wF primer, 10 μM (5′-GAAACTYAAAKGAATTGRCGG-3′; IDT) 1392R primer, 10 μM (5′-ACGGGCGGTGTGTRC-3′; IDT) ExoSAP-IT (Affymetrix, cat. no. 78201) EQUIPMENT Sample preparation and preservation Microcentrifuge tubes, 2 ml (e.g., Eppendorf Safe-Lock tubes 2.0 ml, clear; Eppendorf, cat. no. 0030 120.094) Microcentrifuge (e.g., Eppendorf 5424 ventilated microcentrifuge; Eppendorf, cat. no. 5424 000.410) Vortex (VWR Scientific, Vortex Genie 2) Centrifuge (Eppendorf, cat. no. 5810R) Standard light microscope (Zeiss) Sterile cotton tip (Fisher Scientific, Fisherbrand cotton-tipped applicators, cat. no. 23-400-114) Ultrasonic water bath (e.g., Spectralab ultrasonic cleaning bath; Spectralab Instruments, cat. no. UCB-30D) Falcon tube, 50 ml (Thermo Scientific, cat. no. 362697) Glass beads, 2 mm diameter (e.g., solid-glass beads, borosilicate, diameter 2 mm; Sigma-Aldrich, cat. no. Z273627-1EA) Filter with a 40-μm pore size (e.g., BD Falcon cell strainer 40 μm; BD Biosciences, cat. no. 352340) Filter, 0.2 μm (e.g., Millipore Millex-FG syringe filter unit, 0.2 μm; Millipore, cat. no. SLFG025LS) Flask, 250 ml (Pyrex, cat. no. 4980) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • .. Cell sorter: we use the Influx (BD Biosciences) or MoFlo (Beckman Coulter) with a 70-nm nozzle and 488-nm excitation laser to detect and sort prokaryotic cells labeled with the DNA stain SYBR green.
Sample Prep:Article Title: Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics
Article Snippet: .. STOP solution (Qiagen, cat. no. 1032393) Nuclease-free water (Fisher Scientific, cat. no. BP2484-100) Random hexamers, 50-μM (Integrated DNA Technologies (IDT); see Reagent Setup) DMSO (Sigma-Aldrich, cat. no. D8418-100ML) SYTO 13, 5 mM (Invitrogen, cat. no. S7575) DTT, 1 M (Sigma-Aldrich, cat. no. 646563-10X.5ML; see Reagent Setup) Household bleach (3–8% (wt/vol) solution of sodium hypochlorite) Phylogenetic screening Ultrapure water, such as Milli-Q water (Millipore) or other filtered and deionized water (DIW) SsoAdvanced SYBR Green Supermix (Bio-Rad, cat. no. 172-5264) 926wF primer, 10 μM (5′-GAAACTYAAAKGAATTGRCGG-3′; IDT) 1392R primer, 10 μM (5′-ACGGGCGGTGTGTRC-3′; IDT) ExoSAP-IT (Affymetrix, cat. no. 78201) EQUIPMENT Sample preparation and preservation Microcentrifuge tubes, 2 ml (e.g., Eppendorf Safe-Lock tubes 2.0 ml, clear; Eppendorf, cat. no. 0030 120.094) Microcentrifuge (e.g., Eppendorf 5424 ventilated microcentrifuge; Eppendorf, cat. no. 5424 000.410) Vortex (VWR Scientific, Vortex Genie 2) Centrifuge (Eppendorf, cat. no. 5810R) Standard light microscope (Zeiss) Sterile cotton tip (Fisher Scientific, Fisherbrand cotton-tipped applicators, cat. no. 23-400-114) Ultrasonic water bath (e.g., Spectralab ultrasonic cleaning bath; Spectralab Instruments, cat. no. UCB-30D) Falcon tube, 50 ml (Thermo Scientific, cat. no. 362697) Glass beads, 2 mm diameter (e.g., solid-glass beads, borosilicate, diameter 2 mm; Sigma-Aldrich, cat. no. Z273627-1EA) Filter with a 40-μm pore size (e.g., BD Falcon cell strainer 40 μm; BD Biosciences, cat. no. 352340) Filter, 0.2 μm (e.g., Millipore Millex-FG syringe filter unit, 0.2 μm; Millipore, cat. no. SLFG025LS) Flask, 250 ml (Pyrex, cat. no. 4980) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • .. Cell sorter: we use the Influx (BD Biosciences) or MoFlo (Beckman Coulter) with a 70-nm nozzle and 488-nm excitation laser to detect and sort prokaryotic cells labeled with the DNA stain SYBR green.
Preserving:Article Title: Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics
Article Snippet: .. STOP solution (Qiagen, cat. no. 1032393) Nuclease-free water (Fisher Scientific, cat. no. BP2484-100) Random hexamers, 50-μM (Integrated DNA Technologies (IDT); see Reagent Setup) DMSO (Sigma-Aldrich, cat. no. D8418-100ML) SYTO 13, 5 mM (Invitrogen, cat. no. S7575) DTT, 1 M (Sigma-Aldrich, cat. no. 646563-10X.5ML; see Reagent Setup) Household bleach (3–8% (wt/vol) solution of sodium hypochlorite) Phylogenetic screening Ultrapure water, such as Milli-Q water (Millipore) or other filtered and deionized water (DIW) SsoAdvanced SYBR Green Supermix (Bio-Rad, cat. no. 172-5264) 926wF primer, 10 μM (5′-GAAACTYAAAKGAATTGRCGG-3′; IDT) 1392R primer, 10 μM (5′-ACGGGCGGTGTGTRC-3′; IDT) ExoSAP-IT (Affymetrix, cat. no. 78201) EQUIPMENT Sample preparation and preservation Microcentrifuge tubes, 2 ml (e.g., Eppendorf Safe-Lock tubes 2.0 ml, clear; Eppendorf, cat. no. 0030 120.094) Microcentrifuge (e.g., Eppendorf 5424 ventilated microcentrifuge; Eppendorf, cat. no. 5424 000.410) Vortex (VWR Scientific, Vortex Genie 2) Centrifuge (Eppendorf, cat. no. 5810R) Standard light microscope (Zeiss) Sterile cotton tip (Fisher Scientific, Fisherbrand cotton-tipped applicators, cat. no. 23-400-114) Ultrasonic water bath (e.g., Spectralab ultrasonic cleaning bath; Spectralab Instruments, cat. no. UCB-30D) Falcon tube, 50 ml (Thermo Scientific, cat. no. 362697) Glass beads, 2 mm diameter (e.g., solid-glass beads, borosilicate, diameter 2 mm; Sigma-Aldrich, cat. no. Z273627-1EA) Filter with a 40-μm pore size (e.g., BD Falcon cell strainer 40 μm; BD Biosciences, cat. no. 352340) Filter, 0.2 μm (e.g., Millipore Millex-FG syringe filter unit, 0.2 μm; Millipore, cat. no. SLFG025LS) Flask, 250 ml (Pyrex, cat. no. 4980) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • .. Cell sorter: we use the Influx (BD Biosciences) or MoFlo (Beckman Coulter) with a 70-nm nozzle and 488-nm excitation laser to detect and sort prokaryotic cells labeled with the DNA stain SYBR green.
Light Microscopy:Article Title: Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics
Article Snippet: .. STOP solution (Qiagen, cat. no. 1032393) Nuclease-free water (Fisher Scientific, cat. no. BP2484-100) Random hexamers, 50-μM (Integrated DNA Technologies (IDT); see Reagent Setup) DMSO (Sigma-Aldrich, cat. no. D8418-100ML) SYTO 13, 5 mM (Invitrogen, cat. no. S7575) DTT, 1 M (Sigma-Aldrich, cat. no. 646563-10X.5ML; see Reagent Setup) Household bleach (3–8% (wt/vol) solution of sodium hypochlorite) Phylogenetic screening Ultrapure water, such as Milli-Q water (Millipore) or other filtered and deionized water (DIW) SsoAdvanced SYBR Green Supermix (Bio-Rad, cat. no. 172-5264) 926wF primer, 10 μM (5′-GAAACTYAAAKGAATTGRCGG-3′; IDT) 1392R primer, 10 μM (5′-ACGGGCGGTGTGTRC-3′; IDT) ExoSAP-IT (Affymetrix, cat. no. 78201) EQUIPMENT Sample preparation and preservation Microcentrifuge tubes, 2 ml (e.g., Eppendorf Safe-Lock tubes 2.0 ml, clear; Eppendorf, cat. no. 0030 120.094) Microcentrifuge (e.g., Eppendorf 5424 ventilated microcentrifuge; Eppendorf, cat. no. 5424 000.410) Vortex (VWR Scientific, Vortex Genie 2) Centrifuge (Eppendorf, cat. no. 5810R) Standard light microscope (Zeiss) Sterile cotton tip (Fisher Scientific, Fisherbrand cotton-tipped applicators, cat. no. 23-400-114) Ultrasonic water bath (e.g., Spectralab ultrasonic cleaning bath; Spectralab Instruments, cat. no. UCB-30D) Falcon tube, 50 ml (Thermo Scientific, cat. no. 362697) Glass beads, 2 mm diameter (e.g., solid-glass beads, borosilicate, diameter 2 mm; Sigma-Aldrich, cat. no. Z273627-1EA) Filter with a 40-μm pore size (e.g., BD Falcon cell strainer 40 μm; BD Biosciences, cat. no. 352340) Filter, 0.2 μm (e.g., Millipore Millex-FG syringe filter unit, 0.2 μm; Millipore, cat. no. SLFG025LS) Flask, 250 ml (Pyrex, cat. no. 4980) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • .. Cell sorter: we use the Influx (BD Biosciences) or MoFlo (Beckman Coulter) with a 70-nm nozzle and 488-nm excitation laser to detect and sort prokaryotic cells labeled with the DNA stain SYBR green.
Sterility:Article Title: Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics
Article Snippet: .. STOP solution (Qiagen, cat. no. 1032393) Nuclease-free water (Fisher Scientific, cat. no. BP2484-100) Random hexamers, 50-μM (Integrated DNA Technologies (IDT); see Reagent Setup) DMSO (Sigma-Aldrich, cat. no. D8418-100ML) SYTO 13, 5 mM (Invitrogen, cat. no. S7575) DTT, 1 M (Sigma-Aldrich, cat. no. 646563-10X.5ML; see Reagent Setup) Household bleach (3–8% (wt/vol) solution of sodium hypochlorite) Phylogenetic screening Ultrapure water, such as Milli-Q water (Millipore) or other filtered and deionized water (DIW) SsoAdvanced SYBR Green Supermix (Bio-Rad, cat. no. 172-5264) 926wF primer, 10 μM (5′-GAAACTYAAAKGAATTGRCGG-3′; IDT) 1392R primer, 10 μM (5′-ACGGGCGGTGTGTRC-3′; IDT) ExoSAP-IT (Affymetrix, cat. no. 78201) EQUIPMENT Sample preparation and preservation Microcentrifuge tubes, 2 ml (e.g., Eppendorf Safe-Lock tubes 2.0 ml, clear; Eppendorf, cat. no. 0030 120.094) Microcentrifuge (e.g., Eppendorf 5424 ventilated microcentrifuge; Eppendorf, cat. no. 5424 000.410) Vortex (VWR Scientific, Vortex Genie 2) Centrifuge (Eppendorf, cat. no. 5810R) Standard light microscope (Zeiss) Sterile cotton tip (Fisher Scientific, Fisherbrand cotton-tipped applicators, cat. no. 23-400-114) Ultrasonic water bath (e.g., Spectralab ultrasonic cleaning bath; Spectralab Instruments, cat. no. UCB-30D) Falcon tube, 50 ml (Thermo Scientific, cat. no. 362697) Glass beads, 2 mm diameter (e.g., solid-glass beads, borosilicate, diameter 2 mm; Sigma-Aldrich, cat. no. Z273627-1EA) Filter with a 40-μm pore size (e.g., BD Falcon cell strainer 40 μm; BD Biosciences, cat. no. 352340) Filter, 0.2 μm (e.g., Millipore Millex-FG syringe filter unit, 0.2 μm; Millipore, cat. no. SLFG025LS) Flask, 250 ml (Pyrex, cat. no. 4980) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • .. Cell sorter: we use the Influx (BD Biosciences) or MoFlo (Beckman Coulter) with a 70-nm nozzle and 488-nm excitation laser to detect and sort prokaryotic cells labeled with the DNA stain SYBR green.
Pore Size:Article Title: Obtaining genomes from uncultivated environmental microorganisms using FACS–based single-cell genomics
Article Snippet: .. STOP solution (Qiagen, cat. no. 1032393) Nuclease-free water (Fisher Scientific, cat. no. BP2484-100) Random hexamers, 50-μM (Integrated DNA Technologies (IDT); see Reagent Setup) DMSO (Sigma-Aldrich, cat. no. D8418-100ML) SYTO 13, 5 mM (Invitrogen, cat. no. S7575) DTT, 1 M (Sigma-Aldrich, cat. no. 646563-10X.5ML; see Reagent Setup) Household bleach (3–8% (wt/vol) solution of sodium hypochlorite) Phylogenetic screening Ultrapure water, such as Milli-Q water (Millipore) or other filtered and deionized water (DIW) SsoAdvanced SYBR Green Supermix (Bio-Rad, cat. no. 172-5264) 926wF primer, 10 μM (5′-GAAACTYAAAKGAATTGRCGG-3′; IDT) 1392R primer, 10 μM (5′-ACGGGCGGTGTGTRC-3′; IDT) ExoSAP-IT (Affymetrix, cat. no. 78201) EQUIPMENT Sample preparation and preservation Microcentrifuge tubes, 2 ml (e.g., Eppendorf Safe-Lock tubes 2.0 ml, clear; Eppendorf, cat. no. 0030 120.094) Microcentrifuge (e.g., Eppendorf 5424 ventilated microcentrifuge; Eppendorf, cat. no. 5424 000.410) Vortex (VWR Scientific, Vortex Genie 2) Centrifuge (Eppendorf, cat. no. 5810R) Standard light microscope (Zeiss) Sterile cotton tip (Fisher Scientific, Fisherbrand cotton-tipped applicators, cat. no. 23-400-114) Ultrasonic water bath (e.g., Spectralab ultrasonic cleaning bath; Spectralab Instruments, cat. no. UCB-30D) Falcon tube, 50 ml (Thermo Scientific, cat. no. 362697) Glass beads, 2 mm diameter (e.g., solid-glass beads, borosilicate, diameter 2 mm; Sigma-Aldrich, cat. no. Z273627-1EA) Filter with a 40-μm pore size (e.g., BD Falcon cell strainer 40 μm; BD Biosciences, cat. no. 352340) Filter, 0.2 μm (e.g., Millipore Millex-FG syringe filter unit, 0.2 μm; Millipore, cat. no. SLFG025LS) Flask, 250 ml (Pyrex, cat. no. 4980) • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • • .. Cell sorter: we use the Influx (BD Biosciences) or MoFlo (Beckman Coulter) with a 70-nm nozzle and 488-nm excitation laser to detect and sort prokaryotic cells labeled with the DNA stain SYBR green.
Polymerase Chain Reaction:Article Title: Molecular Data Reveal a Cryptic Diversity in the Genus Urotricha (Alveolata, Ciliophora, Prostomatida), a Key Player in Freshwater Lakes, With Remarks on Morphology, Food Preferences, and Distribution
Article Snippet: After adding 3 μL of D2 buffer (Qiagen) and flicking, the tubes were incubated for 10 min at 65°C in a Mastercycler (Thermal Mastercycler ® nexus gradient, Eppendorf). .. Afterward, 3 μL of the Stop Solution (Qiagen) and 40 μL of the Mastermix (9 μL H 2 O, 29 μL REPLI-g ® Reaction Buffer, 2 μL REPLI-g ® DNA Polymerase) were added into the PCR-tube. ..
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