Total RNA was extracted from 293T WT and Drosha-KO cells. The 3′ terminus of total RNAs were ligated with the irCLIP adaptor by T4 RNA Ligase 1 (NEB, M0437M). After ligation, the RNAs were annealed with a cDNA synthesis primer and cDNAs were generated by SuperScript IV Reverse Transcriptase (Invitrogen, 18090050). The cDNA/RNA duplexes were then purified by streptavidin beads (Invitrogen, 65601) and the RNAs hydrolyzed by NaOH. The cDNA was circularized by CircLigase II ssDNA Ligase (Epicentre, CL9025K). Next, cDNA of snaR-A was amplified from circular single-stranded DNA templates by PCR using primers (P6 tall, P3 tall) that anneal to the irCLIP adaptor and the gene specific reverse primers (snaR-A 5′, snaR-A 3′). The PCR amplicons were purified by Zymoclean Gel DNA Recovery Kit (ZYMO RESEARCH, D4008) and phosphorylated at the 5′ termini by T4 Polynucleotide Kinase (NEB, M0201L). The pBluescript plasmid was cut by EcoRV and dephosphorylated by Calf Intestinal Alkaline Phosphatase (CIP) (NEB, M0290L), then ligated with the phosphorylated PCR fragments by T4 DNA Ligase (NEB, M0202S). Inserts were analyzed by Sanger sequencing. All primers used are listed in Supplemental Table S1 .
Circligase 2 ssdna ligase
Manufactured by Illumina
Sourced in United States
CircLigase II ssDNA Ligase is a thermostable enzyme used for the ligation of single-stranded DNA (ssDNA) molecules. It catalyzes the formation of a phosphodiester bond between the 5' phosphate and the 3' hydroxyl termini of ssDNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin beads, by Thermo Fisher Scientific (2 mentions)
Calf intestinal alkaline phosphatase cip, by New England Biolabs (2 mentions)
T4 rna ligase 1, by New England Biolabs (2 mentions)
Superscript 4 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
T4 polynucleotide kinase, by New England Biolabs (2 mentions)
T4 dna ligase, by New England Biolabs (2 mentions)
Zymoclean gel dna recovery kit, by Zymo Research (2 mentions)
Circligase 2, by Illumina (2 mentions)
Isothermal amplification buffer, by New England Biolabs (1 mentions)
Exonuclease 1, by New England Biolabs (1 mentions)
Templiphi amplification kit, by GE Healthcare (1 mentions)
Rnase r buffer, by Illumina (1 mentions)
Rnase r, by Illumina (1 mentions)
Oligo clean concentrator, by Zymo Research (1 mentions)
Betaine, by Merck Group (1 mentions)
8 protocols using circligase 2 ssdna ligase
1
Cloning and Sequencing of snaR-A
2
Synthetic DNA Nanocluster Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA NCs for loading the Cas12a/crRNA RNP were synthesized by RCA. Sequences of the primer and templates were shown in table S1, where sequences with complete or partial complementation to the target crRNA were incorporated to adjust the interaction between the DNA NCs and Cas12a/crRNA RNP. A palindromic sequence was incorporated to induce the self-assembly of the DNA NC. The templates were 5′-phosphorylated linear single-stranded DNA ordered from IDT. It was cyclized with CircLigase II ssDNA ligase (Epicenter), and the remaining ssDNA was removed by treating the template DNA with Exonuclease I (NEB). After heat inactivation of Exonuclease I, the template (10 pmol) was hybridized with the primer (0.5 μM) in a 1-ml isothermal amplification buffer (NEB) under 95°C for 5 min. After cooling the mixture to room temperature, Bst 2.0 DNA polymerase was added (0.2 U/μl) and the RCA reaction was conducted under 60°C for 17 hours. Precipitates from the reaction were removed by centrifugation at 14,000g for 2 min, and the obtained DNA NC was dialyzed against deionized water in a Slide-A-Lyzer dialysis unit (20-kDa molecular weight cutoff) for 48 hours. Purity and concentration of the synthesized DNA NCs were analyzed using NanoDrop 2000c. Agarose gel electrophoresis (0.8%) was also performed to analyze the DNA NCs.
3
Generation of Pre-Selection Libraries
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Generation of pre-selection libraries for in vitro high-throughput specificity profiling experiments were performed as previously described40 (link). Briefly, 10 pmol of WAS or EMX1 lib oligonucleotides were circularized through incubation with 100 units of CircLigase II ssDNA Ligase (Epicenter) in a total reaction volume of 20 µL for 16 h at 60 °C in 1× CircLigase II Reaction Buffer. The reaction was heat inactivated by incubation at 85 °C for 10 min. 5 pmol of the crude circular ssDNA was converted into concatemeric pre-selection libraries with the illustra TempliPhi Amplification Kit (GE Healthcare) according to the manufacturer’s protocol. Concatemeric pre-selection libraries were quantified with the Qubit 2.0 Fluorometer. Sequences used to generate in vitro pre-selection libraries are listed in Supplementary Table 9.
4
CircLigase II Circularization of RNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA, including synthetic RNA or clipped RNA were circularized with CircLigase™ II ssDNA Ligase (Epicentre) at 60°C for 1 h in a 20 μl reaction volume containing 2 μl 10× reaction buffer, 1 μl 50 mM MnCl2 (Epicentre), 4 μl 5 M Betaine (Epicentre) and 1 μl ligase. To remove the remaining linear RNA, 2.3 μl of 10× RNase R buffer (Epicentre) and 1 μl of RNase R (20 U, Epicentre) was added to the reaction mixture. The RNase R digestion was carried out at 37°C for 10 min. After the digestion, an oligo purification column (Zymo Research, Oligo Clean & Concentrator) was used to isolate the circularized RNA following the manufacturer's instructions. Purified RNA was eluted with nuclease-free water.
5
Oligonucleotide Labeling and Substrate Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All oligonucleotides were PAGE purified where necessary (Eurofins MWG Operon). With the exception of the primer extension oligonucleotide, all oligonucleotides used in this study had an OriL specific sequence (see Supplementary Table S1 for sequence details). Oligonucleotides were radiolabelled on the 5′ or 3′-end. For 5′-end labelling, T4 polynucleotide kinase (NEB) and [γ-32P] ATP were used. For 3′-end labelling, a 51 nt long 26RNA:25DNA oligonucleotide was radiolabelled to generate a 52 nt long 26RNA:26DNA oligonucleotide by one of two methods: 3′-end labelling using Klenow fragment fill-in and [α-32P] dCTP; or, 3′-end labelling using terminal transferase (NEB) and [α-32P] dCTP, followed by 3′ overhang removal using T4 DNA polymerase (NEB). For assays using a minicircle substrate, a 120 nt template oligonucleotide was circularized using CircLigase II ssDNA ligase (Epicenter). Labelling occurred by the incorporation of [α-32P] dCTP during the experimental reactions. Further details about substrates are given in the assay-specific sections below.
6
Circular Ligation of cDNA Fragments
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ethanol precipitated cDNA reaction product (see above) was resuspended in 10.0 μl circular ligation reaction mix containing 1.0 μl 10× reaction buffer (0.33M Tris-acetate pH 7.8, 0.66M potassium acetate, 5 mM DTT), 2.5 mM MnCl2, 1M betaine and 1.25 units CircLigase II ssDNA Ligase (Epicentre). Reactions were incubated at 60°C for 2 h.
7
Optimized CircLigase II Ligation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
1 to 6 μg (∼10 to 60 pmol) of DNA was incubated with 2.5 mM MnCl2, 1 M betaine (Sigma, St. Louis, MO, USA), and 50 to 100 U CircLigaseII ssDNA ligase (Epicenter, Madison, WI, USA) in 1× CircLigaseII reaction buffer at 60°C for 16 h, followed by heat inactivation of the ligase at 80°C for 10 min. Highest conversion yields were observed when ligating 6 μg DNA with 100 U CircLigase.
8
Isolation and Cloning of snaR-A
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from HEK293T WT and Drosh-KO cells. The 3' terminus of total RNAs were ligated with the irCLIP adaptor by T4 RNA Ligase 1 (NEB, M0437M). After ligation, the RNAs were annealed with a cDNA synthesis primer and cDNAs were generated by SuperScript™ IV Reverse Transcriptase (Invitrogen, 18090050). The cDNA/RNA duplexes were then purified by streptavidin beads (Invitrogen, 65601) and the RNAs hydrolyzed by NaOH. The cDNA was circularized by CircLigase™ II ssDNA Ligase (Epicentre, CL9025K). Next, cDNA of snaR-A was amplified from circular single-stranded DNA templates by PCR using primers (P6 (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
tall, P3 tall) that anneal to the irCLIP adaptor and the gene specific reverse primers (snaR-A 5', snaR-A 3'). The PCR amplicons were purified by Zymoclean™ Gel DNA Recovery Kit (ZYMO RESEARCH, D4008) and phosphorylated at the 5' termini by T4 Polynucleotide Kinase (NEB, M0201L). The pBluescript plasmid was cut by EcoRV and dephosphorylated by Calf Intestinal Alkaline Phosphatase (CIP) (NEB, M0290L), then ligated with the phosphorylated PCR fragments by T4 DNA Ligase (NEB, M0202S). Inserts were analyzed by Sanger sequencing. All primers used are listed in Supplemental Table S1.
tall, P3 tall) that anneal to the irCLIP adaptor and the gene specific reverse primers (snaR-A 5', snaR-A 3'). The PCR amplicons were purified by Zymoclean™ Gel DNA Recovery Kit (ZYMO RESEARCH, D4008) and phosphorylated at the 5' termini by T4 Polynucleotide Kinase (NEB, M0201L). The pBluescript plasmid was cut by EcoRV and dephosphorylated by Calf Intestinal Alkaline Phosphatase (CIP) (NEB, M0290L), then ligated with the phosphorylated PCR fragments by T4 DNA Ligase (NEB, M0202S). Inserts were analyzed by Sanger sequencing. All primers used are listed in Supplemental Table S1.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!