PP7-based mRNP purification was performed as previously described (Hogg and Goff 2010 (link)). For SDS-PAGE, complexes were eluted with RNase A/T1 Cocktail (AM2286; Ambion), diluted 1:50 in HLB150, as previously described (Hogg and Goff 2010 (link)). For mass spectrometry, samples were eluted in HLB supplemented with 1 M NaCl. For isolation of protein-stripped mRNAs, PP7-based mRNP purifications were performed and mRNA phenol-extracted using TRIzol (15596026; Invitrogen). Capped and polyadenylated in vitro transcribed NLuc RNA was prepared using ApaI-linearized NLuc template DNA and the mMessage mMachine T7 Transcription (AM1344; Ambion) and Poly(A) Tailing (AM1350; Ambion) kits according to manufacturer instructions. NLucβwtβTP and NLucβwtSMG5TP IVT mRNAs were prepared using PCR-amplified cDNA as a template for the in vitro transcription reactions (PCR primers: 5′ Forward: TAA TAC GAC TCA CTA TAG GGA GAC; 3′ Reverse: AGG AAA GGA CAG TGG GAG TG).
Rnase a t1 cocktail
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
RNase A/T1 Cocktail is a ready-to-use solution containing a mixture of RNase A and RNase T1 enzymes. It is designed for the digestion of single-stranded RNA in various molecular biology applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (1 mentions)
Optiprep, by Merck Group (1 mentions)
Proteinase k, by Roche (1 mentions)
Dynabeads m 270 epoxy, by Thermo Fisher Scientific (1 mentions)
Las 4000 imager, by Fujifilm (1 mentions)
Ecl select reagent, by GE Healthcare (1 mentions)
Goat anti mouse igg hrp, by Santa Cruz Biotechnology (1 mentions)
Supersignal west pico chemiluminescent substrate, by Thermo Fisher Scientific (1 mentions)
Las 4000, by Cytiva (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Oligo dt cellulose, by Thermo Fisher Scientific (1 mentions)
Recombinant ifn γ, by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
S1 nuclease, by Thermo Fisher Scientific (1 mentions)
N acetyl cysteine (nac), by Merck Group (1 mentions)
Nigericin, by Merck Group (1 mentions)
Sorafenib, by Merck Group (1 mentions)
Z devd fmk, by Santa Cruz Biotechnology (1 mentions)
Mcc950, by InvivoGen (1 mentions)
Tlrl picw, by Merck Group (1 mentions)
11 protocols using rnase a t1 cocktail
1
Purification and Analysis of mRNPs
2
Purification of Recombinant Virus-Like Particles
3
Genomic DNA Extraction Protocol
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To extract genomic DNA, cells were resuspended in genomic lysis buffer (300 mM NaCl, 1% SDS, 20 mM EDTA) and incubated with Proteinase K (Roche) at 55 °C overnight. RNA was removed by incubation with RNase A/T1 Cocktail (Ambion) at 37 °C for 1 h, in between two phenol–chloroform extraction steps. DNA was quantified by Nanodrop 8000 spectrophotometer and purity was assessed by electrophoresis.
4
Immunoprecipitation and Western Blotting Assay
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CoIPs were performed essentially as described in the previous section with the following modifications: 50 ODs of cells were grown at 30°C in EMM 0.5X and harvested by centrifugation. Cell pellets were resuspended in 2 ml lysis buffer to make ‘pop-corn’. 1 mg of pre-washed rabbit IgG-conjugated M-270 Epoxy Dynabeads (Invitrogen, #14311D) was used for immunoprecpitation in the presence or the absence of RNaseA/T1 cocktail (Ambion, #AM2286). Following washes, precipitates and input fractions were boiled at 95°C for 10 min in the presence of sample loading buffer and analyzed by SDS-PAGE and Western blotting using 1:3000 peroxydase-conjugated antiperoxydase (PAP) (Sigma, #P1291, RRID:AB_1079562 ), 1:3000 monoclonal anti-FLAG antibody (Sigma, #F3165, RRID:AB_259529 ), 1:3000 monoclonal anti-GFP antibody (Roche, #11814460001, RRID:AB_390913 ) and 1:4000 goat anti-mouse IgG-HRP (Santa Cruz Biotechnology, #sc-2005, RRID:AB_631736 ). Detection was performed using SuperSignal West Pico Chemiluminescent Substrate (ThermoFischer Scientific, #34080), ECL Select reagent (GE Healthcare, #RPN2235) and a Fujifilm LAS-4000 imager.
5
Quantifying DNA Methylation by HPLC
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Genomic DNA was extracted using DNeasy Blood and Tissue kit (Qiagen) following manufacturer's protocol or standard phenol chloroform precipitation and eluted in water. DNA was treated with RNase A/T1 Cocktail (Ambion) overnight at 37°C followed by ethanol precipitation. Quantitation of 5mC in genomic DNA was done by isocratic high performance reverse phase liquid chromatography (HPLC) as previously described (28 (link)) with the following alterations. A Dionex UM 3000 HPLC system was used complete with a column chiller, C18 column (250 mm × 4.6 mm 5 μM APEX ODS, Grace Discovery Sciences), and column guard (Phomenex). The mobile phase was 50 mM ammomium phosphate (monobasic) pH4.1. The column was chilled to 8°C to improve peak separation. Deoxyribonucleotides (dNMPs) were detected at their extinction maxima using a Dionex 3000 multiple wavelength detector: dCMP, 276 nm; 5mdCMP, 282 nm. Quantifications were calculated from the area under each peak using the respective extinction coefficients (dCMP, 8.86 × 103; 5mdCMP 9.0 × 103).
6
Rapamycin-Induced mRNA Capture Protocol
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Cells were stimulated with rapamycin for 24 h and extracts prepared in lysis buffer as described above. Oligo dT cellulose (Ambion) was resuspended in lysis buffer twice before incubating it in the presence of cell extracts on a rotating wheel at 4°C during 2 h. Cellulose-bound extracts were washed five times. RNAse treatment was done using 1/100 RNAse A/T1 cocktail (Ambion) at 37°C during 13 min shaking. Extracts were washed once more and cellulose was pelleted and resuspended in lysis buffer, boiled and loaded onto bis/acrylamide gels for CBP80/eIF4E blotting.
7
Immune Activation Protocol Reagents
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The following chemicals and drugs were utilized in this study: Talabostat/VbP (MCE #HY-13233A), Lipofectamine 2000 (Invitrogen #11668019), double-stranded alternating copolymer poly(dA:dT) (pdAdT) (Sigma #P0883), poly(I:C) (pIC) (Invivogen #tlrl-picw), HT-DNA (Sigma #D6898), double-stranded homopolymer poly(dA):poly(dT) (Sigma #P9764), poly(dG:dC) (Invivogen #tlrl-pgcn), PAM3CSK4 (Invivogen #tlrl-pms), nigericin (Sigma #N7143), diABZI (MCE #HY-112921B), ANS (MCE #HY-18982), H2O2 (Sigma #H1009), MG-132 (MCE #HY-13259), Bortezomib (MCE #HY-10227), MCC950 (Invivogen #inh-mcc), z-VAD-FMK (Santa Cruz #sc-3067), z-DEVD-FMK (Santa Cruz #sc-311558), H-151 (MCE #HY-112693), NAC (Sigma #A9165), KU-44933 (Santa Cruz #sc-202963), NU-7441 (Tocris #3712), Sorafenib (Sigma #SML2633), PLX-4720 (MCE #HY-51424), Doramapimod (MCE #HY-10320), SB-202190 (MCE #HY-10295), and RNA Polymerase III inhibitor (Sigma #557403). gDNA was isolated from the genomic DNA of HEK293T cells. ISD was synthesized from custom oligos as previously described (55 (link)). Recombinant IFNγ was purchased from Peprotech (#300-02). DNase I (Bio-Rad #7326828), S1 Nuclease (Thermofisher #EN0321), and RNase A/T1 Cocktail (Thermofisher #AM2286) were purchased from the indicated vendors. VACV Copenhagen strain WT and ΔF1L were a kind gift of John Bell (56 (link)) and titered by plaque assay.
8
Optimizing RNase A/T1 for RNA Duplex Enrichment
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RNase A/T1 cocktail (500 U/mL RNase A and 20,000 U/mL RNase T1) (ThermoFisher Scientific, Waltham, MA, USA) was used for enriching RNA duplexes. For optimization, 1 μg of annealed oligos were digested in 150 μL of digestion buffer (300 mM NaCl, 10 mM Tris–Cl, 5 mM ethylenediaminetetraacetic acid (EDTA), pH 7.4) with different concentrations of RNase A/T1 (1:100, 1:200, and 1:500) at 37 °C for 30 min. For optimizing the RNase A/T1 condition in the context of cellular RNA, 1 × 106 cells were lysed on ice with 50 μL of radioimmunoprecipitation assay (RIPA) buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) (ThermoFisher Scientific, Waltham, MA, USA). After adding different annealed RNA oligos as spike-in, cell lysates were digested in an additional 100 μL of digestion buffer with RNase A/T1 (1:200) at 37 °C for 30 min. RNA was separated on 15% acrylamide Tris/borate/EDTA (TBE) (K-D Medical, Columbia, MD, USA) native gels and stained with SYBR gold (ThermoFisher Scientific, Waltham, MA, USA). Images were obtained with Chemidoc Imaging system (Bio-Rad, Hercules, CA, USA).
9
Transfer of Synthetic sRNA from Astrocytes
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To confirm that the synthetic sRNA was transferred from astrocyte cytoplasm, a co-culture experiment was carried out as described above. Prior to co-culturing, astrocytes-22bpCy3 were treated for 10 min at 37°C with RNase A/T1 cocktail (0.4 μg/μl, Thermo Scientific) to degrade any synthetic sRNA residues attached to astrocyte membranes, as described elsewhere [39 (link)]. Transfer of 22bpCy3 to PC14 cells was examined by FACS as described above.
10
Drosophila S2R+ Cell Transfection and RNase Treatment
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Drosophila S2R+ cells were plated in 6-well plates at a density of 5.106 cells/well and incubated for 24 hr at 25°C. Cells were then transfected with 600 ng of plasmids using Effecten transfection reagents (Qiagen, 301425) and supplemented after 12 hr with Schneider’s Insect Medium containing fetal bovine serum (10%) and penicillin/streptomycin (1%). After 3 days of expression at 25°C, cells transfected with either pAGW + pAFlag-CamkII or pAGFP-Imp + pAFlag-CamkII plasmids were harvested and lysed in DXB buffer (HEPES pH 6.8, 2.5 mM; KCl, 50 mM; MgCl2, 1 mM; DTT, 1 mM; sucrose, 250 mM; NP-40, 0.05%) supplemented with Halt Protease Inhibitor Cocktail 1:100 (Thermofisher, #78429). This initial lysate was used as input material and then split into two halves, one treated (+ RNase) with 10 mg/µL RNase A/T1 Cocktail (ThermoFisher scientific, #EN0551) and the other not (− RNase). Both cell lysates were subsequently incubated with ChromoTek GFP-Trap beads (ChromoTek, gt-10, #70112001A) for 2 hr at 4°C. Three washes were then performed with DBX supplemented with Halt Protease Inhibitor Cocktail 1:100, and bound fractions eluted in SDS 2× buffer by incubating the beads at 95°C for 5 min.
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