RNase R treatment and circRNA quantification was performed according to a published protocol (Panda and Gorospe, 2018) with minor modification. In brief, RNA was treated with 20 μl RNase R digestion reaction (2 μg RNA, 1 μl RNase R (Lucigen, #RNR07250), 2 μl 10× RNase R reaction buffer) and control reaction without RNase R. The reactions were incubated for 30 min at 37°C and immediately purified using ZYMO RNA Clean & Concentrator (ZYMO RESEARCH, D7011). The purified RNA samples were eluted in 20 μl of nuclease free water, and 12 μl of RNase R treated RNA and control RNA were used for reverse transcription. Quantification of circRNAs were performed using iTaq Universal SYBR Green Supermix (BioRad, #1725124) according to the manufacturer's instructions. The qPCR reactions were prepared as follows: 0.1 μl cDNA, 10 μl of SYBR Green Supermix, 1.2 μl primer mix (5 μM each) and 6.8 μl nuclease-free water. Reactions were carried on CFX Connect Real-Time PCR Detection System for 2 min at 95°C and 40 cycles of 5 s at 95°C and 20 s at 60°C followed by melting curve analysis. The enrichment of RNA after RNase R treatment was calculated using the delta CT method, mouse gene Gapdh and Rps14 were used as linear control.
Zymo rna clean concentrator
Manufactured by Zymo Research
Sourced in United States
The Zymo RNA Clean & Concentrator is a laboratory equipment designed to purify and concentrate RNA samples. It utilizes a silica-based spin column technology to efficiently remove contaminants and concentrate RNA samples for downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Bsa blocked brdu beads, by Santa Cruz Biotechnology (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Rnase r, by Zymo Research (1 mentions)
Itaq universal sybr green supermix, by Bio-Rad (1 mentions)
Hme dip kits, by Active Motif (1 mentions)
Dneasy blood and tissue dna kit, by Qiagen (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Ez dna methylation gold kit, by Zymo Research (1 mentions)
Rnase free dnase 1, by Illumina (1 mentions)
Agilent bioanalyzer, by Agilent Technologies (1 mentions)
Tapestation agilent instrument, by Agilent Technologies (1 mentions)
Rneasy plus universal kit, by Qiagen (1 mentions)
6 protocols using zymo rna clean concentrator
1
Quantifying Circular RNAs by RNase R Digestion
2
Epigenomic Profiling of Srap1 in Mouse ESCs
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For ChIP experiments, mouse ESCs stably expressing FHA-Srap1 were fixed in 1% paraformaldehyde for 10 min at 37 C, followed by quenching with 125 mM glycine for 5 min. Cells were lysed and processed using the MAGnify ChIP system (Life Sciences). For DIP, genomic DNA was isolated from Srap1 KO and matched wild-type ESCs using the DNeasy Blood and Tissue DNA kit (QIAGEN) and sonicated for a total of 6 min. Samples were processed using the MeDIP or hMeDIP kits (Active Motif) according to the manufacturer's protocol. For RNA-seq, total RNA was extracted using the Zymo RNA Clean & Concentrator (Zymo Research), followed by rRNA depletion and cDNA preparation using the KAPA Stranded RNA-Seq Kit with RiboErase. For RRBS, genomic DNA was extracted from E9.5 wild-type and Srap1 KO embryos using the DNeasy Blood and Tissue kit and digested with MspI overnight. Sizeselected (<0.7 kb) DNA fragments were subjected to bisulfite conversion using the EZ DNA Methylation-Gold kit (Zymo Research), followed by 12 cycles of PCR amplification using Kapa HiFi HotStart Uracil+ polymerase. Indexed libraries were prepared using the Kapa HyperPrep kit and purified with 0.93 Ampure XP beads (Beckman Coulter), pooled, and analyzed on an Illumina Hi-Seq2000 or NextSeq500.
3
Nascent RNA Analysis by Global Run-on
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Analysis of nascent RNAs using global-run-on experiments were performed as described previously57 . Briefly, nuclei were isolated in swelling buffer (10mM Tris-HCl pH7.5, 2mM MgCl2, 3mM CaCl2), lysed twice in lysis buffer (10mM Tris-HCl pH7.5, 2mM MgCl2, 3mM CaCl2, 10% glycerol, 0.5% NP-40) and snap-frozen in freezing buffer (50mM Tris pH8.0, 40% glycerol, 5mM MgCl2, 0.1mM EDTA), For run-on reaction, an equal volume of reaction buffer was added to thawed nuclei (10mM Tris pH8.0, 5mM MgCl2, 300mM KCl, 500uM ATP, 500µM GTP, 5µM CTP, 500µM BrUTP, 1mM DTT, 100U/mL SuperaseIN, 1% Sarcosyl), mixed and incubated at 30°C for 5min. The reaction was stopped with Trizol reagent and RNA was phenol/chloroform extracted and ethanol precipitatated. RNA was heated in Fragmentation buffer (40mM Tris pH8.0, 100mM KCl, 6.25mM MgCl2, 1mM DTT), DNAse treated and purified using Zymo RNA Clean & Concentrator (Zymo Research) using the >17nt protocol. Run-on RNA was immunoprecipitated using BSA-blocked BrDU beads (Santa Cruz) in Binding buffer (SSPE 0.5X, 1mM EDTA, 0.05% Tween-20) for 1h at 4°C, washed and eluted in Elution buffer (5mM Tris pH7.5, 300mM NaCl, 20mM DTT, 1mM EDTA, 1% SDS) at 65°C for 20min. Nascent RNA was further phenol/chloroform extracted and sequencing libraries were prepared.
4
RNA Extraction from C. jejuni Mutants
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C. jejuni 11168 wild type, ΔpseC and ΔpseF mutant cells were harvested from overnight NZCYM plate cultures, pelleted and washed once in NZCYM broth and set to an OD600 of 0.05 (2 × 108 colony forming units per mL (CFU/mL)) in 20 mL NZCYM broth in 125-mL Erlenmeyer flasks, each containing a 1-inch sterile magnetic stir-bar. Cells were grown under microaerobic conditions and magnetically stirred at 200 rpm. After 4.5 h incubation (mid-log phase, cell counts were approximately 5 × 108 CFU/mL), the entire contents of each flask was transferred to a pre-prepared tube containing 2.6 mL (0.1 volume) ice cold 10% buffered phenol in 100% ethanol to stabilize RNA followed by immediate mixing and storage on ice until all samples were collected [37 (link)]. RNA was extracted from each sample using a hot phenol method [37 (link)]. RNA samples were sequentially DNAse-treated (37 °C for 30 min) using RNAse-free DNAse I (Epicentre, Madison, WI, USA) and cleaned using the Zymo RNA Clean & Concentrator (Zymo Research, Irvine, CA, USA). PCR was used to confirm the absence of residual DNA. Total RNA quality was assessed using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA) and RNA was stored at −80 °C until further use. Samples were extracted in biological triplicate.
5
Liver Transcriptome Analysis of Cattle
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From the frozen liver samples, a total of 36 samples (12 from each breed) consisting of six samples from animals with extreme high and six extreme low RFI phenotypes from each of the three breeds were selected for total RNA extraction and consequently differential gene expression analyses. The frozen liver tissue of each steer was pulverised into fine powder using liquid nitrogen with a pre-chilled mortar and pestle on dry ice. Total RNA was then extracted from 10 mg of the pulverised tissue using a Qiagen RNeasy Plus Universal Kit (Qiagen, Toronto, ON, Canada) and further purified using a Zymo RNA Clean & Concentrator (Zymo, Irvine, CA, USA). RNA was quantified using a NanoDrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA) and was deemed acceptable if its absorbance (A260/280) was between 1.8 and 2.0. RNA integrity was confirmed using a TapeStation-Agilent instrument (Agilent Technologies, Mississauga, ON, Canada), and the RNA integrity number (RIN) values for all samples were higher than 8.
6
Nascent RNA Analysis by Global Run-on
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