About 5 μg fragmented mRNA was subjected to immunoprecipitation, according to the reported MeRIP method [16 (link)]. Briefly, fragmented RNA was incubated for 2 h at 4°C with 10 μg m6A antibody (Synaptic Systems Cat. No.202003, diluted to 0.5 mg/ ml) in 1,000 μl RIP buffer (50 mM Tris-HCl, 750 mM NaCl and 0.5% Igepal CA-630), supplemented with 2 mM RVC (Sigma) and 200 U RNasin (Promega). To reduce nonspecific binding, protein-A beads were pre-blocked in 1,000μl RIP buffer with 0.5 mg/ml BSA for 2h at 4°C. The pre-blocked protein-A beads were then incubated with above mixture for another 2 h at 4°C. The beads were vigorously washed using 1,000 μl RIP buffer three to four times. Discard the RIP buffer. Add 300 μl dilution buffer (10 mM Tris-HCl pH 7.5) into the bead tube and incubate at 50°C for 90 min. Eluted RNA was precipitated by ethanol-NaAc solution and glycogen (Life Technologies) overnight at -80°C. The eluted RNA was treated with RNasin (Promega) according to the manufacturer’s instructions.
Rnasin
Manufactured by Promega
Sourced in United States, Germany, United Kingdom, France, Switzerland, Sweden, Japan
RNasin is a recombinant ribonuclease inhibitor protein that inhibits RNase activity. It is used to protect RNA from degradation during in vitro procedures.
Automatically generated - may contain errors
Lab products found in correlation
Trizol, by Thermo Fisher Scientific (56 mentions)
Trizol reagent, by Thermo Fisher Scientific (49 mentions)
Rneasy mini kit, by Qiagen (27 mentions)
Dithiothreitol (dtt), by Thermo Fisher Scientific (27 mentions)
M mlv reverse transcriptase, by Promega (26 mentions)
Rq1 dnase, by Promega (20 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (20 mentions)
Dntps, by Thermo Fisher Scientific (20 mentions)
Protease inhibitor cocktail, by Roche (18 mentions)
Superscript 3, by Thermo Fisher Scientific (17 mentions)
T7 rna polymerase, by Promega (16 mentions)
Dnase 1, by Thermo Fisher Scientific (16 mentions)
Sw41ti rotor, by Beckman Coulter (14 mentions)
Amv reverse transcriptase, by Promega (14 mentions)
Cycloheximide (chx), by Merck Group (13 mentions)
Dntps, by Promega (13 mentions)
M mlv reverse transcriptase, by Thermo Fisher Scientific (13 mentions)
Rnaseout, by Thermo Fisher Scientific (12 mentions)
Protease inhibitor cocktail, by Merck Group (11 mentions)
Dnase 1, by Promega (11 mentions)
776 protocols using rnasin
1
Isolation of m6A-Containing mRNA
2
ASCC3 Immunoprecipitation in HEK293T Cells
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ASCC3 immunoprecipitation was carried out using HEK293T cells. 50–60% confluent HEK293T cells in a 10 cm dish were treated overnight with 0.05% DMSO or 7.5 μM PF8503. Cells were washed 3 times with HBSS buffer (Gibco, 14025092) and detached by scraping in 1 mL HBSS. Cell pellets were collected after centrifugation and resuspended in 200 μL IP lysis buffer (25 mM Tris-HCl pH 7.4, 150 mM KCl, 0.5 mM DTT, 1X Protease inhibitor, Sigma-Aldrich 4693116001, 4 mM EDTA, 0.5% NP40, 100 U/mL RNAsin, Promega N2511), incubated 10 min on ice, and centrifuged at 4°C for 10 min (14000xg) to remove cell debris. Total cell lysates were incubated with 1 μg of ASCC3 antibody (Bethyl laboratories, A304-015A) for 1 hr at 4°C on a rotator. Then 40 μL of cell lysate was stored at -20°C as the input fraction of Western blot gels. The remaining 160 μL was mixed with 50 μL protein G beads that had been washed and resuspended in 50 μL of NT2 buffer (25 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM MgCl2, 1X protease inhibitor, Sigma-Aldrich 4693116001, 0.05% NP40, and 100U/mL RNAsin, Promega N2511) and the samples were incubated overnight at 4°C on a rotator. Protein G beads were washed 4 times with NT2 buffer. Proteins bound to the beads were eluted by denaturation in 1X NuPage buffer at 95°C for 10 min and protein content of each fraction were analyzed by Western blot, along with the loading control.
3
Purification of Orb-containing RNPs
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Mouse anti-Orb (4H8 and 6H4) or mouse anti- β-gal (401A) were coupled to A/G agarose beads (Santa Cruz Biotechnology) by incubating overnight at 4 degrees. 250 females were dissected in ice cold 1xPBS and ovaries were transferred to dry ice while dissecting. RNAsin (Promega) was added to ovaries and they were crushed using a plastic pestle to make a paste. The ovary paste was centrifuged at 3000 rpm for 5 minutes at 4 degrees twice, and the supernatant was saved. Half of the supernatant was added to the Orb antibody coupled with beads, and the other half was added to the control antibody beads. CoIP buffer ([55 (link)]) and RNAsin (Promega) was added to IPs, which were left to rotate for 3 hours at 4 degrees. The beads were pelleted by centrifugation and washed with coIP buffer 5 times.
RNA was released from the beads by adding 10 mM HEPES 1% SDS solution and β-mercaptoethanol, and left in a 65 degree water bath for 15 minutes. Phenol followed by phenol chloroform was used for extraction, and the water phase was ethanol precipitated with glycogen added as a carrier. The pellet was dried and then DNAse (Promega) treated.
RNA was released from the beads by adding 10 mM HEPES 1% SDS solution and β-mercaptoethanol, and left in a 65 degree water bath for 15 minutes. Phenol followed by phenol chloroform was used for extraction, and the water phase was ethanol precipitated with glycogen added as a carrier. The pellet was dried and then DNAse (Promega) treated.
4
Zebrafish Polysome Profiling
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150 embryos per sample were collected at 3 dpf and identified by phenotype. Embryos were deyolked and rinsed with ice cold PBS and then dissociated in ice-cold lysis buffer (10mM Tris-HCl, 5 mM MgCl2, 100 mM KCL, 1% TritonX-100, 2 mM DTT, 100ug/ml cycloheximide, 200U/ml RNasin (Promega), and protease inhibitor (Sigma)). Homogenized zebrafish were then centrifuged at 15000 x g at 4°C for 10 minutes and the supernatant of the zebrafish lysate was kept for analysis. Lysates were loaded onto a 10–50% sucrose gradient prepared in 20 mM HEPES-KOH pH 7.4, 5 mM MgCl2, 100 mM KCl, 2mM DTT, 20 U/mL RNasin (Promega), and 100 ug/mL cycloheximide. The gradients were ultra-centrifuged at 4°C in an SW-41 Ti rotor (Beckman) at 40,000 rpm for 2 hours. To evaluate UV absorbance profiles, each gradient was passed through a UA-6 absorbance reader system (Teledyne ISCO) using a syringe pump (Brandel). The absorbance at 254 nm was recorded using WinDaq data acquisition software (DATAQ INSTRUMENTS) and the profiles were plotted in Microsoft Excel.
5
Single-cell cDNA Synthesis Protocol
6
Isolation and Quantification of A. suum Intestinal Nuclei
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Prior to extracting nuclei, we assessed numbers of nuclei that comprise the A. suum intestine. This was accomplished by staining freshly dissected intestine with the non-vital stain bisbenzimide (Hoechst nuclear dye 33,258, Sigma-Aldrich, St. Louis MO) at 10 µg/mL in PBS for 30 min. Rinsed tissue placed on a glass slide. The intestine assumed a flattened shape, rather than round. It was then viewed using a Nikon Diaphot 300 inverted microscope equipped with epifluorescence capabilities (UV-2 A filter (blue) and photographed with a Nikon D5100 digital camera. Digital images were evaluated by counting the average number of nuclei contained within 50µM × 50µM areas of intestinal tissue, and extrapolating the cell counts to the full intestine.
To obtain single nuclei preparations, flash frozen A. suum intestinal tissue (∼ 50 mg) was homogenized in a Dounce homogenizer in 3 mL Lysis Buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.025% NP-40, and 0.04 U/µL RNasin (Promega)) and incubated on ice 15 min. The suspension was filtered through a 30 μm filter to remove debris and pelleted at 500 × g for 5 min at 4oC. Nuclei were washed and filtered twice with Nuclei Wash (1% BSA in PBS with 0.2 U/µL RNasin (Promega)). The nuclei pellets were resuspended in Nuclei Wash at 1000 nuclei / µL and filtered through a 40 μm FlowMi Cell Strainer.
To obtain single nuclei preparations, flash frozen A. suum intestinal tissue (∼ 50 mg) was homogenized in a Dounce homogenizer in 3 mL Lysis Buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.025% NP-40, and 0.04 U/µL RNasin (Promega)) and incubated on ice 15 min. The suspension was filtered through a 30 μm filter to remove debris and pelleted at 500 × g for 5 min at 4oC. Nuclei were washed and filtered twice with Nuclei Wash (1% BSA in PBS with 0.2 U/µL RNasin (Promega)). The nuclei pellets were resuspended in Nuclei Wash at 1000 nuclei / µL and filtered through a 40 μm FlowMi Cell Strainer.
7
RNA to cDNA Conversion Protocol
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Two microgram of total RNA were supplemented with 5 µL of a master mix: 1 µL oligo dT (8 nmol) (Roche Diagnostic, Meylan, France), 4 µL of RNAse/DNAse free water and 0.1 µL RNAsin (40U/µL, Promega, charbonnières, France). Then, samples were incubated at 70°C for 10 min, followed by 5 min at room temperature. After this 10 µL of the reaction mix were added to samples: 6 µL buffer 5X (Tris‐HCL, KCL, MgCL2) (Invitrogen, UK) + 1 µL DiThioThreitol (DTT, 0.1 M) (Invitrogen, UK) + 2 µL deoxyribose Nucleotide Triphosphates (dNTPs, 10 mM) (Amersham Biosciences, UK) + 0.1 µL RNAsin (40 U/µL, Promega, charbonnières, France) + 1 µL Transcriptase Reverse Superscript (200 U/µL, life technologies, Carlsbad, CA, USA). Samples were first incubated at 45°C for 60 min, followed by a second incubation of 5 min at 95°C. Finally, ultrapure distilled water (GIBCO‐Life Technologies) was added to obtain a final concentration of 10 ng total complementary DNA (cDNA)/µL and stored at −80°C until further use.
8
RNA-Protein Interaction Profiling
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The RNA binding protein pull-down assay was performed as described [35 (link)]. Briefly, 30 μg of in vitro synthetic RNAs were denatured at 65°C for 5min, and then applied to 50 μl streptavidin beads (Thermo) in 800 μllysis buffer (RIPA lysis buffer, Beyotime) with protease inhibitor (Roche) and RNasin (200 U/ml, Promega) after cooling to room temperature. The mixer were incubated on a rotation shaker at 4°C for 2–2.5 h. Total mouse brain lysate was collected by centrifuging at 4°C for 10 min at 16,000 g after grinded in lysis buffer (RIPA lysis buffer, Beyotime) with protease inhibitor (Roche) and RNasin (200 U/ml, Promega). The supernatants were incubated with Yeast RNA (Sigma) and Egg white avidin (EMD chemicals) to block endogenous biotinylated proteins and non-specific RNPs, and were cleared by centrifuge. Next, the cleared lysates were incubated with RNA-beads in a new tube on a rotation shaker at 4°C for 2.5–3 h. The supernatant was discarded after centrifugation for 2 min at 4°C. After washing with fresh lysis buffer for 5 times, the captured proteins were eluted and analyzed by SDS–PAGE with silver staining. The specific RNA binding protein bands were cut from the gel and subjected tocommercial mass spectrometry analysis (Shanghai applied protein technology, China).
9
PAPAS-Mediated Genomic DNA Enrichment
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Genomic DNA was fragmented by sonication and digestion with AccI and SalI to an average size of 200–300 bp. DNA (7.5 µg) was incubated with 20 pmol of in vitro transcribed PAPAS (Supplemental Table S1, S2 ) for 40 min at 37°C in triplex buffer (10 mM Tris-HCl at pH 7.4, 50 mM KCl, 5 mM MgCl2) supplemented with 0.8 U/µL RNasin (Promega). DNA–RNA complexes were bound to MyOne Streptavidin C1 Dynabeads (Invitrogen). After three washes with triplex buffer containing 0.05% Tween20 and 0.5% NP-40 and one wash with triplex buffer containing 0.05% Tween20, RNA-associated DNA was eluted with 25 ng/µL RNase and 5 mU/µL RNase 1 (Thermo Scientific) for 30 min at 37°C. DNA was recovered by phenol/chloroform extraction and analyzed by qPCR.
To monitor binding of PAPAS to defined DNA fragments, PCR fragments were generated using GoTaq DNA polymerase (Promega). Four-hundred femtomoles of exonuclease I-treated PCR fragments was incubated with 20 pmol of biotinylated PAPAS for 1 h at 37°C in triplex buffer containing 10 mM Tris-HCl (pH 7.4), 50 mM KCl, 5 mM MgCl2, 0.8 U/µL RNasin (Promega), and 10 µg of salmon sperm DNA (Invitrogen). After binding to streptavidin beads, washing, and elution, PAPAS-associated DNA was recovered by phenol/chloroform extraction and analyzed by qPCR.
To monitor binding of PAPAS to defined DNA fragments, PCR fragments were generated using GoTaq DNA polymerase (Promega). Four-hundred femtomoles of exonuclease I-treated PCR fragments was incubated with 20 pmol of biotinylated PAPAS for 1 h at 37°C in triplex buffer containing 10 mM Tris-HCl (pH 7.4), 50 mM KCl, 5 mM MgCl2, 0.8 U/µL RNasin (Promega), and 10 µg of salmon sperm DNA (Invitrogen). After binding to streptavidin beads, washing, and elution, PAPAS-associated DNA was recovered by phenol/chloroform extraction and analyzed by qPCR.
10
In Vitro Viral RNA Synthesis Assay
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