To analyze Triton X-100 soluble/insoluble SAF-A, cells were treated with CSK buffer containing 100 mM NaCl, 0.1% Triton X-100, 300 mM Sucrose, 1 mM MgCl2, 1 mM EGTA, 10 mM PIPES (pH 6.8), and 100 μM PMSF (phenylmethylsulfonyl fluoride) on ice for 10 min, followed by immuno-staining or divided into the supernatant and pellet fractions by centrifugation, at 5000 rpm, 4°C for 5 min, for western blotting. To analyze DNase or RNase A/T1 extraction of SAF-A, cells were treated with CSK buffer containing 100 units.mL-1 DNase I (ThermoFisher) and 2.5 mM CaCl2 or 0.5/20 units.ml-1 RNase A/T1 (Ambion) for 30 min at R.T. and were divided into the supernatant and pellet by centrifugation, at 5000 rpm, 4°C for 5 min.
Rnase a t1
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
RNase A/T1 is a ribonuclease enzyme used in molecular biology applications. It is a mixture of RNase A and RNase T1, which are both endoribonucleases that specifically cleave single-stranded RNA at different sites. RNase A cleaves after pyrimidine nucleotides, while RNase T1 cleaves after guanine nucleotides.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase k, by Thermo Fisher Scientific (5 mentions)
Tri reagent, by Merck Group (3 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Rnase t1, by Thermo Fisher Scientific (2 mentions)
Superase in rnase inhibitor, by Thermo Fisher Scientific (2 mentions)
Rnase a, by Thermo Fisher Scientific (2 mentions)
Maxiscript kit, by Thermo Fisher Scientific (2 mentions)
Tnt coupled reticulocyte lysate system with sp6 rna polymerase, by Promega (2 mentions)
Uhplc 1290 instrument, by Agilent Technologies (2 mentions)
Agilent 1290 dad, by Agilent Technologies (2 mentions)
Genejet genomic dna extraction kit, by Thermo Fisher Scientific (2 mentions)
Gfp trap ma beads, by Proteintech (2 mentions)
Triton x 100, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (2 mentions)
Hitrap chelating hp column, by GE Healthcare (2 mentions)
Hitrap heparin hp column, by GE Healthcare (2 mentions)
Hiload 16 600 superdex 200 column, by GE Healthcare (2 mentions)
Hitrap, by Cytiva (2 mentions)
Mini quick spin dna column, by Roche (2 mentions)
50 protocols using rnase a t1
1
Triton X-100 Fractionation of SAF-A
2
Isolation and Enrichment of Autophagosomes
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Autophagosomes were fractionated as described30 (link). Briefly, 20 160 cm2 plates of HEK293 cells were harvested in 0.25 M sucrose 10 mM HEPES pH 7.2, washed several times in the same solution, transferred into 0.25 M sucrose containing protease inhibitor cocktail (Roche, EDTA-free), and disrupted by nitrogen cavitation (Parr Instruments, 100 p.s.i. 30 s, 50 p.s.i. 30 s, and 25 p.s.i. 8 min). Lysate was centrifuged twice at 2000× g to eliminate intact cells and large debris. Supernatant was then centrifuged at 17,000× g (12 min) to pellet organelles. The pellet was resuspended in 0.25 M sucrose 10 mM HEPES, pH 7.2, and treated with a mixture of RNAse A/T1 (Fermentas) for 10 min, 37 °C. Organelles were then loaded at the bottom of a discontinuous Histodenz density gradient (26, 24, 20, and 15%) and centrifuged at 90,000× g for 3 h in an SW41 rotor (Beckman). Fractions at the interface of 15–20% and 20–24% densities are enriched in autophagosomes and autophagolysosomes, respectively. Autophagosome-enriched fractions were used for all analyses.
3
RNA-Protein Interaction Footprinting
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Proteins (10 pmol each) were mixed with 5 pmol 32P body‐labeled RNA to a final 20 μl reaction volume in 50 mM HEPES pH 6.5, 150 mM NaCl, 1 mM magnesium diacetate, 10% (v/v) glycerol, 0.1% (w/v) NP‐40, and 1 mM DTT. After incubation for 1 h at 4°C, the reaction mixtures were digested with 1 μg RNase A/T1 mix and 2.5 U RNase T1 (Fermentas) for 20 min at 20°C. Protected RNA fragments were then extracted twice with phenol:chloroform:isoamyl alcohol (25:24:1 (v/v), Invitrogen), precipitated with ethanol, separated on a denaturing 22% (w/v) polyacrylamide gel, and visualized by phosphorimaging (Fuji).
4
Quantitative Analysis of DNA Methylation Patterns
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TLC was performed as previously described (Ficz et al., 2011 (link); Tahiliani et al., 2009 (link)). 2 μg of genomic DNA isolated from control, Dnmt1 KD, Mbd3 KD, or Mbd2 KD ES cells was digested overnight with 50 units of MspI (NEB) and 10 μg of RNaseA/T1 (Ambion) at 37°C. The reaction was then heat inactivated at 65°C, DNA was dephosphorylated with 10 units of rSAP (NEB) for 1 hr at 37°C and cleaned over a DNA clean and concentrator column (Zymo Research, Irvine, CA, USA). 400 ng of DNA was incubated with 10 μCi 32P-ATP and 10 U of T4 PNK for 1 hr at 37°C. Labeled DNA was precipitated, resuspended in 18 μL 30 mM Tris pH 8.9, 15 mM MgCl2, 2 mM CaCl, and fragmented to single nucleotides with 10 units of DNaseI (NEB) and 10 μg SVPD (Worthington, Lakewood, NJ, USA) for 3 hr at 37°C. 3 μL of samples were spotted onto PEI cellulose F TLC plates (Millipore) and developed in isobutyric acid:H2O:NH3 (66:20:1 v/v/v) for 15 hr. Plates were dried and exposed to film for 30 hr. Plate was then exposed to a phosphorimager screen for 110 hr and scanned on a Typhoon FLA 700 (GE Healthcare). Pixels were quantitated using Fiji ImageJ software.
5
RNAP II Inhibition and Kinetics
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To inhibit transcription by RNAP II, N2A cells were treated with 10 μg/mL α-amanitin or the indicated concentrations of DRB for the indicated periods of time. For slowing the RNAP II speed, N2A cells were transfected with WTS, C4, or α-amanitin-resistant WTR RNAP II. α-Amanitin (10 μg/mL) was added to the medium to inhibit endogenous RNAP II at 24 h after transfection, and the cells were incubated for an additional 24 h. The cells were lysed in NETN buffer. For RNase treatments, total cell lysates were incubated with 1 µL of RNase A/T1 (Ambion) for 20 min at 30°C. Cleared cell lysate was incubated with 2 μg of the indicated antibody to form an immune complex for 2 h at 4°C. Next, 20 μL of Dynabeads protein G was added and incubated for 1 h at 4°C. Immune complexes were washed four times by NETN buffer and analyzed by Western blotting.
6
Ribosome Profiling in Liver Tissue
7
Characterization of RNA-Protein Interactions
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Preparation of RNA substrates and myc-tagged proteins were carried out as described previously21 (link). In brief, the RNA substrates were synthesized from PCR-generated DNA templates by T7 RNA polymerase in the presence of [α-32P]UTP using a MAXIscript kit (Ambion). The myc-tagged proteins were synthesized in vitro from pCS3+MT constructs using the TNT Coupled Reticulocyte Lysate System with SP6 RNA polymerase (Promega). Binding reactions were carried out in a 25 μl mixture that contains 10 mM HEPES (pH 7.9), 2 mM MgCl2, 1 mM ATP, 20 mM creatine phosphate, 50 ng yeast tRNA, 2 mM DTT, 2% polyethylene glycol (M.W. 3,550), the reticulocyte lysate reaction mixture and 1 × 106 c.p.m. RNA probe for 20 min at 30 °C. Reaction mixtures were irradiated with 254 nm UV in a UV Stratalinker 2400 (Stratagene) for 20 min on ice, digested with 2 μl RNase-A/T1 (Ambion) and immunoprecipitated with 2 μg of anti-myc. Samples were subject to LDS-PAGE followed by autoradiography. The radioactive bands were quantified using NIH ImageJ.
8
Immunoprecipitation of Protein Complexes
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Before IP, the cells were washed with PBS and lysed in IP buffer (50 mM HEPES [pH 7.5], 150 mM NaCl, 1 mM EDTA, 2.5 mM EGTA, 10% glycerol, 0.1% NP-40) supplemented with a protease inhibitor cocktail (Roche). After brief sonication the lysates were cleared by centrifugation (10,000g) at 4°C for 10 min and treated with RNase A/T1 (Ambion) for 10 min at room temperature. Prewashed, antibody-coated beads (Sigma or Millipore) were added to the cell lysate and incubated overnight at 4°C. After extensive washes with IP buffer the immunoprecipitated complexes were eluted with IP buffer supplemented with 150 µg/mL of 3×Flag peptide (Sigma) for 2 h at 4°C or by 2× SDS protein sample buffer supplemented with 5% (vol/vol) 2-mercaptoethanol. Immunoprecipitated proteins were separated in SDS-PAGE and analyzed by mass spectrometry or Western blot.
9
Characterization of RNA-Protein Interactions
10
RNA Extraction and Analysis Protocol
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