Kidneys were perfused, minced, and placed in DMEM (Life Technologies, Carlsbad, CA, USA) containing 1 mg/ml collagenase IA and 100 ng/ml RNAse I (Sigma-Aldrich, St. Louis, MO, USA) at 37°C for 30 min. Digested kidney tissues were passed through a 40-μm cell strainer (BD Falcon, San Diego, CA, USA), and the cell suspension obtained was centrifuged at 300 × g for 10 min. Cells were washed in PBS containing 2% BSA, suspended in 36% Percoll (Amersham Pharmacia Biotech, Little Chalfont, UK), and gently overlaid onto 72% Percoll. After centrifugation at 900 × g for 30 min at RT, cells were retrieved from the Percoll interface and washed twice in DMEM and once with staining buffer (PBS containing 2% BSA and 0.1% sodium azide).
Rnase 1
Manufactured by Merck Group
Sourced in United States
RNase I is an enzyme that catalyzes the hydrolysis of single-stranded RNA. It is a laboratory reagent used for the degradation of contaminating RNA in various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Propidium iodide, by Merck Group (6 mentions)
Dnase 1, by Merck Group (5 mentions)
Flow cytometry, by BD (3 mentions)
Nuclease p1, by Merck Group (3 mentions)
Proteinase k, by Merck Group (3 mentions)
Alkaline phosphatase, by Merck Group (3 mentions)
Calibur, by BD (2 mentions)
Summit 4, by Agilent Technologies (2 mentions)
Facscanto 2, by BD (2 mentions)
Facsaria system, by BD (2 mentions)
Ez magna rip rna binding protein immunoprecipitation kit, by Merck Group (2 mentions)
Amicon micropure ez filter, by Merck Group (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Percoll, by Cytiva (1 mentions)
40 μm cell strainer, by BD (1 mentions)
Collagenase ia, by Merck Group (1 mentions)
Accuri c6, by BD (1 mentions)
Anti m6a antibody, by Abcam (1 mentions)
Magna rip rna binding protein immunoprecipitation kit, by Merck Group (1 mentions)
Novaseq 6000 platform, by Illumina (1 mentions)
31 protocols using rnase 1
1
Kidney Cell Isolation Protocol
2
Cell Cycle Analysis by Flow Cytometry
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To analyze cell cycle distribution, 1 × 106 cells were harvested and washed with PBS, then resuspended in 1 ml PBS. The cells were fixed in 9 ml ice cold 75% ethanol at 4°C overnight and then washed with PBS and resuspended in propidium iodide (20 μg/ml in PBS with 0.1% Triton-X 100, Sigma Aldrich) and RNaseI (100 μg/ml, Sigma Aldrich). Samples were incubated at 37°C in water bath for 15 minutes and analyzed by flow cytometer (Accuri C6, BD Biosciences).
3
Validating siRNA Protection by MSNPs
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For validation of the protective effect of MSNPs to the linked siRNA, the siRNA linked MSNPs or free siRNA targeting different mRNAs were incubated separately with 0.25% (w/v) RNase I (Sigma-Aldrich, Poole, UK) for different periods of time, ranging from 15 min to 4 h (for free siRNA) or even longer, up to 24 h. Gel electrophoresis was used to assess degradation as above. As previously, 2% (w/v) native agarose gel electrophoresis was used to assess degradation for 1 h at 100 V. The gel was prepared under the same conditions.
4
Cell Cycle Analysis of HUVECs
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A total of 2×106 HUVECs/ml were collected by centrifugation (1,000 × g; 5 min; 4°C) and washed twice with PBS buffer, prior to being fixed with ice-cold 70% ethanol for 5 min at 4°C. The cells were incubated with 1 µg/ml RNase I (Sigma-Aldrich; Merck KGaA) at 37°C for 1 h in the dark. Following the addition of 20 µg/ml propidium iodide at 37°C for 15 min, the samples were analyzed using an LSR II flow cytometer (BD Biosciences). Cells were analyzed using FlowJo version 10.0 software (FlowJo LLC).
5
m6A Profiling of Bladder Cancer Cells
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For the m6A RNA binding experiments, the RNAs of bladder cancer cells stably transfected with either the lentiviral overexpression or control ALKBH5 vector were isolated and treated with RNase I (Sigma-Aldrich, USA). RNAs were fragmented by sonication for 10 s on an ice-water mixture. Immunoprecipitations were performed using an anti-m6A antibody (1:1,000; Abcam, USA) previously bound to magnetic Life Technologies Dynabeads (Thermo Fisher Scientific, USA) in the RIPA buffer (Magna RIP RNA-Binding Protein Immunoprecipitation Kit; Millipore Sigma, USA) and incubated with DNA-free fragmented RNAs. Beads were then treated with proteinase K (20 mg/mL) for 1.5 h at 42°C. Subsequently, RNAs were extracted using phenol/chloroform/isoamyl alcohol and subjected to qRT-PCR using primers for CK2α and normalized to input.
6
Cell Cycle Analysis by FACS
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Cells were fixed with ice-cold ethanol and re-suspended in PBS containing RNase I (100 mg/mL, Sigma) and propidium iodide (50 mg/mL, Sigma). Samples were subjected to fluorescence-activated cell sorting (FACS, Calibur, Becton Dickinson), and data were analyzed using the Summit 4.3 software (DAKO Cytomation) as previously described (Federico et al., 2016 (link)).
7
Cell Cycle Analysis by Flow Cytometry
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Cultured cells were harvested and washed with PBS to analyze the cell cycle. The collected cells were fixed with 75% alcohol overnight at 4°C and then treated with RNase I (Sigma) for 15 minutes. After washing with PBS, the cells were stained with 50 mg/ml propidium iodide (Sigma). The cells were then immediately subjected to LSRII flow cytometry (Beckman Coulter, Inc., Brea, CA) and analyzed using FlowJo v10.0.8. A minimum of 20 000 cells were analyzed for each sample.
8
DNase and RNase Cleavage Assay for Nucleic Acid-Protein Complex
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For the DNase and RNase cleavage assay, 5 μg of the purified nucleic acid and protein complex was incubated with 2 U of DNase I (Sigma-Aldrich) and RNase I (Sigma-Aldrich) in buffer (25 mM Tris-HCl, pH 8.0, 150 mM NaCl, 3 mM DTT) at 37 °C for 30 min, respectively. The cleavage products were monitored with 10% TBE-urea-PAGE gel electrophoresis and visualized by EB staining.
The IS607 TnpB RNP complex was treated with 2 U of DNase I (Sigma-Aldrich) at 37 °C for 30 min and reRNA was subsequently purified by phenol/chloroform extraction. In brief, small RNA libraries were constructed using the NEB Next Multiplex Small RNA Library Prep Set for Illumina according to the manufacturer’s protocol. The library was sequenced on NovaSeq 6000 platform (Illumina) by Shanghai Personal Biotechnology Co. Ltd. (Shanghai, China).
The IS607 TnpB RNP complex was treated with 2 U of DNase I (Sigma-Aldrich) at 37 °C for 30 min and reRNA was subsequently purified by phenol/chloroform extraction. In brief, small RNA libraries were constructed using the NEB Next Multiplex Small RNA Library Prep Set for Illumina according to the manufacturer’s protocol. The library was sequenced on NovaSeq 6000 platform (Illumina) by Shanghai Personal Biotechnology Co. Ltd. (Shanghai, China).
9
Cell Cycle Analysis by Flow Cytometry
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First, 1 × 106 cells were cultured in a 100-mm dish overnight, followed by treatment with 1 at 10 μg/mL for 24 h. Then, cells were trypsinized and washed with PBS three times. Cells were fixed with 4% paraformaldehyde and 50 μL of 100 μg/mL Rnase I (Sigma-Aldrich, St. Louis, MO, USA) was added to fixed cells and incubated for 1 h at 37 °C. Lastly, 200 μL of 50 μg/mL propidium iodide was added and the DNA contents of the cells were analyzed using flow cytometry (Gallios, Beckman Coulter, Brea, CA, USA).
10
Cell Cycle Analysis by FACS
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Cells were fixed with ice-cold ethanol and resuspended in PBS containing RNase I (100 mg/ml, Sigma) and propidium iodide (50 mg/ml, Sigma). Samples were subjected to fluorescence activated cell sorting (FACS, Calibur, Becton Dickinson), and data was analyzed using the Summit 4.3 software (DAKO Cytomation).
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