The HTR2C and MAGT1 minigenes were cloned into pcDNA3.1 plasmid and cotransfected with Flag only, Flag-ADAR2, CSII-CMV-EV, or CSII-CMV-DAP3 plasmids into EC109 cells. Cells were lysed by prechilled lysis buffer [50 mM tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100 × cOmplete protease inhibitor (Roche), and SUPERase·In RNase Inhibitor (0.1 U/μl) (Invitrogen)] and incubated overnight with M2 magnetic beads at 4°C. Then, the M2 magnetic beads were washed six times with tris-buffered saline buffer [50 mM tris (pH 7.5), 150 mM NaCl, and SUPERase·In RNase Inhibitor (0.02 U/μl) (Invitrogen)]. Bound proteins were eluted with 2× protein loading buffer after boiling at 95°C for 10 min. WB was performed to examine pull-down efficiency. RNAs bound to the M2 magnetic beads were eluted with buffer RLT and purified with the RNeasy Mini Kit (Qiagen). Complementary DNA (cDNA) was synthesized using the Advantage RT-for-PCR Kit (Takara), and quantitative PCR (qPCR) was performed using GoTaq qPCR Master Mix (Promega). Enrichment of pulled-down RNAs was normalized to the input RNA expression levels. Primer sequences can be found in table S4.
Superase in rnase inhibitor
Manufactured by Thermo Fisher Scientific
Sourced in United States
SUPERase-In RNase Inhibitor is a recombinant ribonuclease (RNase) inhibitor protein that binds to and inhibits the activity of RNases. It is designed to protect RNA from degradation during RNA-related experiments and procedures.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (15 mentions)
Turbo dnase, by Thermo Fisher Scientific (13 mentions)
Rnase 1, by Thermo Fisher Scientific (12 mentions)
Trizol reagent, by Thermo Fisher Scientific (11 mentions)
Taqman gene expression assay, by Thermo Fisher Scientific (10 mentions)
Cycloheximide (chx), by Merck Group (9 mentions)
Proteinase k, by Thermo Fisher Scientific (8 mentions)
Cellsdirect 2x reaction mix, by Thermo Fisher Scientific (7 mentions)
Rneasy mini kit, by Qiagen (6 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (6 mentions)
Dnase 1, by Thermo Fisher Scientific (6 mentions)
Rnase a, by Thermo Fisher Scientific (5 mentions)
Rnase t1, by Thermo Fisher Scientific (5 mentions)
Ampure xp bead, by Beckman Coulter (5 mentions)
Triton x 100, by Merck Group (5 mentions)
Maxima h minus reverse transcriptase, by Thermo Fisher Scientific (5 mentions)
Dynabeads protein a, by Thermo Fisher Scientific (5 mentions)
Rnasin plus rnase inhibitor, by Promega (4 mentions)
Itaq universal sybr green supermix, by Bio-Rad (4 mentions)
Rt buffer, by Thermo Fisher Scientific (4 mentions)
290 protocols using superase in rnase inhibitor
1
Studying RNA-Protein Interactions
2
Identifying RNA-binding Protein Interactors
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Cells were lysed by prechilled lysis buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1% Triton, 1×complete protease inhibitor (Roche), and 0.1U/μl SUPERase·In™ RNase Inhibitor (Invitrogen)) and incubated overnight with M2 magnetic beads at 4oC. Then the M2 magnetic beads were washed six times with TBS buffer (50 mM Tris (pH 7.5), 150 mM NaCl, and 0.02U/μl SUPERase·In™ RNase Inhibitor (Invitrogen)). Bound proteins were eluted with 2× protein loading buffer after boiling at 95 °C for 10 min. Western blot was performed to examine pulldown efficiency. RNAs bound to the M2 magnetic beads were eluted with buffer RLT and purified with RNeasy Mini Kit (Qiagen). cDNA was synthesized using SensiFAST™ cDNA synthesis kit (Bioline) and qPCR was performed using GoTaq® qPCR Master Mix (Promega). Enrichment of pulled down RNAs were normalized to the input RNA expression levels. Primer sequences are listed in Supplementary data 7 .
3
RNA Fractionation and Purification Protocol
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Total RNA was isolated from S2 cells and unfractionated embryos using TRIzol® (Invitrogen) and miRVana miRNA isolation kit (Ambion, Waltham, MA, USA), respectively. Ribonucleoprotein complexes were fractionated according to a previously published procedure with minor modifications [22 (link)]. Briefly, embryos (0.2 g) or S2 cells (3 × 107) were homogenized in 5 mL lysis buffer ((100 mM NaCl, 10 mM MgCl2, 30 mM Tris-HCl (pH 7), 1% Triton X-100, 1% NaDOC, 100 µg/mL cycloheximide (Applichem, Darmstadt, Germany) and 30 U/mL SUPERase.In™ RNase Inhibitor (Ambion, Waltham, MA, USA)) and incubated on ice for 8 min. The homogenates were centrifuged at 12,000× g at 4 °C for 8 min. Two-mL supernatant was loaded onto 5–70% (w/v) sucrose gradients (100 mM NaCl, 10 mM MgCl2, 30 mM Tris-HCl (pH 7), 200 U SUPERase. In™ RNase inhibitor (Ambion, Waltham, MA, USA)) and centrifuged at 27,000 rpm for 2 h 55 min at 4 °C in a Beckman SW28 rotor. Fractions were collected using an ISCO density gradient system. Fractions were then pooled into 4 sub-groups based on their A254 readings; mRNP, 60S, monosome and polysome. Total RNA was extracted from the fractions as previously described [22 (link)]. RNA quality was assessed by 2100 bioanalyzer using RNA 6000 Nano Kit based on manufacturer’s instructions (Agilent, Santa Clara, CA, USA).
4
Single-Cell RNA Sequencing Sample Preparation
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Samples were thawed on ice for 2 minutes, then centrifuged at 2,500 rpm at 4°C for 1 minute and the RNA concentration normalized. 1.9 μL of RNA per sample were moved to a full-skirt 96-well plate (Eppendorf). Each sample was then mixed with 1μL 10μM RT primer 5’AAGCAGTGGTATCAACGCAGAGTACTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTVN-3’ (IDT), 1μL 10 mM dNTP (Life Technologies/Thermo Fisher Scientific), and 0.1μL SUPERase•In RNase-Inhibitor (20 U/μL, Life Technologies/Thermo Fisher Scientific). Samples were denatured at 72° C for 3 minutes using an Eppendorf Mastercycler and placed immediately on ice afterwards. 7 μL of the Reverse Transcription Mix was subsequently added to every well, consisting of: 2 μL 5x RT buffer (Thermo Fisher Scientific), 2 μL 5 M Betaine (Sigma-Aldrich), 0.9 μL 100 mM MgCl2 (Sigma-Aldrich), 1 μL 10 μM TSO (5’-AAGCAGTGGTATCAACGCAGAGTACATrGrG+G-3’, Exiqon), 0.25 μL SUPERase•In RNase-Inhibitor (20U/μL, Life Technologies/Thermo Fisher Scientific), 0.1 μL Maxima H Minus Reverse Transcriptase (200U/μL, Thermo Fisher Scientific), and 0.75 μL nuclease-free water. Reverse transcription was carried out by incubating the plate at 50°C for 90 minutes, followed by heat inactivation at 85°C for 5 minutes.
5
Single-Cell RNA Sequencing Sample Preparation
6
Isolation and Purification of Neuronal Nuclei
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Nuclei were isolated from frozen VC tissue. Tissue was manually homogenized using a two-step Dounce homogenizer (A and B) (Sigma #D9063) in NIMT buffer, containing (in mM: 250 sucrose, 25 KCl, 5 MgCl2, 10 Tris-Cl pH 8, 1 DTT; 1:100 dilution of Triton X100, Protease Inhibitor Cocktail [Sigma #P8340]; and 1:1000 dilution of RNaseOUT Recombinant Ribonuclease Inhibitor [Thermo #10777019]; SUPERase• In RNase Inhibitor [Thermo #AM2694]) on ice. Homogenized samples were mixed with 50% iodixanol (OptiPrep Density Gradient Medium; Sigma #D1556) and loaded onto 25% iodixanol cushion, and centrifuged at 10,000 g for 20 min at 4°C in a swinging bucket rotor (Sorval HS-4). Pellets resuspended in ice-cold DPBS (HyClone) with 1:1000 dilution of RNaseOUT Recombinant Ribonuclease Inhibitor (Thermo #10777019); SUPERase• In RNase Inhibitor (Thermo #AM2694). Nuclei were then incubated for 7 min on ice with Hoechst 33342 solution (20 mM) (Thermo #62249) (final concentration 0.5 µM), followed by centrifugation at 1000 g for 10 min at 4°C to pellet nuclei. Pellets were resuspended in blocking buffer containing DPBS with RNAse inhibitors, and 1:10 dilution of pure BSA, and blocked for 30 min on ice. NEUN-Alexa488 pre-conjugated antibody (Millipore #MAB377X) was then added at 1:1000 dilution and incubated for at least 1 hr on ice before proceeding to flow cytometry sorting.
7
Reverse Transcription of ncRNA and mRNA
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cDNA was generated in a total reaction volume of 20 μl using poly(A) tailing-based RT. The RT reaction mix was as follows: 4 μl RT-buffer (5X), 1 μl 2.5 μM poly A adapter/primer (Integrated DNA Technologies, Inc.), 1 μl 5 mM dNTPs (Jena Bioscience), 0.25 μl Maxima™ H Minus reverse transcriptase (Thermo Fisher Scientific, Inc.), 0.25 μl SUPERase In™ RNase inhibitor (Thermo Fisher Scientific, Inc.), 0.5 μl 10 mM ATP (New England BioLabs, Inc.), 0.25 μl poly A polymerase (New England BioLabs, Inc.) and the required amount of RNA sample. Briefly, 1,000 ng of isolated intracellular ncRNA and 50 ng of isolated microvesicular extracellular ncRNA were used for RT. Due to the small amount of ncRNA in serum and urine, a fixed volume of 5 μl was used. RT was performed at 42°C for 30 min and 85°C for 10 min. Processed cDNA was stored at 4°C. Additionally, 2,000 ng of RNA was used for the RT of mRNA. The RT reaction mix contained 5 μl RT-buffer (5X), 1 μl 5 mM dNTPs (Jena Bioscience), 1 μl RT primer (10 μM), 0.25 μl Maxima™ H Minus reverse transcriptase (Thermo Fisher Scientific, Inc.), 0.25 μl SUPERase In™ RNase inhibitor (Thermo Fisher Scientific, Inc.) and the RT was carried out in a total volume of 25 μl. The RT temperature protocol was performed as follows: 65°C for 1 min, 25°C for 10 min, 50°C for 45 min and 85°C for 10 min.
8
Measuring Protein-RNA Binding in Yeast Cells
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Previously described methods with some modifications3 (link) were used to measure the binding of Pop1, Est1 and Est2 to TLC1. Briefly, cells expressing HA-Pop1, Est1-MYC or Est2-MYC were collected from 250 mls of culture grown to OD600nm = 0.5 at 24 °C. Cell lysis was accomplished with glass-beads in TMG100 buffer (10 mM Tris-Cl pH8, 1 mM MgCl2, 10% glycerol, 100 mM NaCl, 0.1 mM EDTA, 0.1 mM DTT). A complete mini EDTA-free protease inhibitor tablet, 20 µls of RNasin Plus RNase Inhibitor (Promega N2618), and 20 µls of SUPERase In RNase Inhibitor (Invitrogen AM2696) were added to 10 mls of TMG100 buffer. For immunoprecipitations, 500 µls of TMG100 plus inhibitors, 0.5% Tween 20 and 10 µls of monoclonal c-MYC antibody (Takara 631206) or anti-HA (Santa Cruz sc-7392) were added to 1 mg of total protein and incubated overnight at 4 °C. Dynabeads protein G (Invitrogen) were equilibrated with TMG100 and 0.5% Tween-20, added to the samples and incubated for 4 h at 4 °C. After three washes with TMG100 and 0.5% Tween-20 and one wash with TMG100, the beads were resuspended in TMG100. The IP and INPUT samples were treated with proteinase K (Thermo Fisher 2546), and RNA was extracted with hot acidic phenol method and ethanol precipitated as described above. RNA samples were DNased with Turbo DNA-free (Invitrogen AM107) and eluted with 50 µls of TE buffer.
9
N6-methyladenine Immunoprecipitation Protocol
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For N6-methyladenine immunoprecipitation (MeRIP) we followed protocols of “Magna MeRIP m6A kit” (Sigma-Aldrich, cat. No. 1710499), Dominissini and Meyer group33 (link),52 (link). 0.125 mg of Pierce Protein A/G Magnetic Beads (Thermofisher, cat. no. 88803) were coupled with 5 µg of m6A antibody (Synaptic Systems, cat. no. 202–003) in 500 μL IP buffer (50 mM Tris–HCl 7.5 pH, 150 mM NaCl, 0.1% (vol/vol) Igepal) for 6 h at 4 °C. Bead-antibody complex was then washed twice with 1 mL IP buffer for 10 min. 5 µg of fragmented polyA RNA was mixed with 500 µL IP buffer (supplemented with 0.3 U/µL SUPERase-In RNase inhibitor (Invitrogen, cat. no. AM2694) and 2 mM RVC) and placed on washed bead-antibody complex for overnight incubation at 4 °C. Mixture was washed twice for 10 min. with 1 mL IP buffer (supplemented with 0.1 U/µL SUPERase-In RNase inhibitor) and once with 1 mL high salt buffer (50 mM Tris–HCl 7.5 pH, 300 mM NaCl, 0.1% (vol/vol) Igepal, 0.1 U/µL SUPERase-In RNase inhibitor). Bead-antibody-RNA complex was treated twice with 100 µL elution buffer (IP buffer supplemented with m6A salt at 6.7 mM (Sigma-Aldrich, cat. no. M2780)) for 1 h at 4 °C. RNA was recovered by overnight ethanol precipitation (2.5 vol 100% ethanol and 1/10 volume of 3 M sodium acetate: pH 5.2) at −80 °C.
10
Single-cell RNA-seq of Planarian Cells
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Dissociated planarian cells stained with the Near-IR LIVE/DEAD Dye and Hoechst 33342 were sorted with an influx sorter using a 100μm tip. Hoechst was excited with 100mW of 355nm UV and collected behind a 450/20 bandpass. Near-IR LIVE/DEAD Dye was excited with 631nm and collected behind a 750LP filter. Sorting was done in purity mode at approximately 12,000 – 15,000 eps. For each sample, 400,000 – 500,000 single Hoechst+/DeadDye− cells were collected for barcoding. Split-pool ligation-based barcoding of dissociated cells was performed as previously described (22 (link)), then centrifuged at 1000g for 10 minutes in a swinging bucket centrifuge and re-suspended in 1X PBS + SUPERase•In RNase Inhibitor (Invitrogen AM2694). All oligonucleotides and primers sequences were ordered from IDT with the sequences previously reported (22 (link)). Single, intact Hoechst+ cells were sorted with an influx sorter using a 100μm tip and 150,000 – 200,000 cells were re-suspended in 400μL 1X PBS + SUPERase•In RNase Inhibitor. Cells were split into 8 sub-libraries, with 50μL of suspension going into each tube. 50μL lysis buffer (20mM Tris, pH 8.0, 400mM NaCl, 100mM EDTA, pH 8.0, 4.4% SDS) and 10μL proteinase K (20mg/mL) was added to the barcoded cells. Lysates were incubated at 55°C for 2 hours with 200rpm agitation, then stored at −70°C until library preparation.
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