RNA with or without 5’ adaptor ligation (see below) was subjected to 3’ adaptor ligation by mixing 12 μl of RNA (<5 μg) with 1 μl of 100 μM 3’ adaptor (Supplementary Table 5 ), 0.5 μl of 50 mM ATP, 2 μl of dimethyl sulfoxide, 5 μl of 50% PEG8000, 1 μl of RNase Inhibitor (New England BioLabs, M0314), and 1 μl of High Concentration T4 RNA Ligase 1 (New England BioLabs, M0437). After incubation at 23 °C for 5 hr, the reaction was diluted to 40 μl with water and purified twice with 1.5× vol of Agencourt RNAClean XP beads (Beckman Coulter, A63987) to remove excess RNA adaptors. The sample was subsequently eluted in 12 μl of water.
T4 rna ligase 1 high concentration
Manufactured by New England Biolabs
T4 RNA Ligase 1 - high concentration is a DNA and RNA modifying enzyme used to join RNA or DNA fragments. It catalyzes the formation of a phosphodiester bond between the 5' phosphate and 3' hydroxyl termini of RNA or DNA. This enzyme is supplied at a high concentration for increased efficiency in ligation reactions.
Automatically generated - may contain errors
Lab products found in correlation
Rnase inhibitor, by New England Biolabs (4 mentions)
Agencourt rnaclean xp beads, by Beckman Coulter (2 mentions)
Agilent high sensitivity dna kit, by Agilent Technologies (2 mentions)
Miseq reagent kit v3, by Illumina (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Phusion high fidelity dna polymerase, by New England Biolabs (2 mentions)
Superscript 4 reverse transcriptase, by Thermo Fisher Scientific (2 mentions)
5 deadenylase, by New England Biolabs (2 mentions)
Rna fragmentation reagent, by Thermo Fisher Scientific (1 mentions)
Rnase h, by New England Biolabs (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Rneasy minelute cleanup kit, by Qiagen (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
Recjf, by New England Biolabs (1 mentions)
Q5 high fidelity 2 master mix, by New England Biolabs (1 mentions)
6 protocols using t4 rna ligase 1 high concentration
1
3' Adapter Ligation of RNA
2
3' Adapter Ligation of RNA
3
Small RNA Library Construction and Sequencing
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5 μg of total RNA was ligated with RNA 3’ adaptor using T4 RNA Ligase 2 - truncated (NEB), in the presence of RNase Inhibitor (NEB). RNA 5’ adaptor was ligated using T4 RNA Ligase 1 - high concentration (NEB) and 10 mM ATP. Ligated small RNAs were reverse transcribed using SuperScript® IV Reverse Transcriptase (Thermo-Fisher). Small RNA library cDNA was amplified and indexed using Phusion® High-Fidelity DNA polymerase (NEB). Constructs were purified in a 6% (w/v) native acrylamide gel based on the expected product size and purified by ethanol precipitation. Library quality was assessed by using Qubit dsDNA HS Assay Kit (ThermoFisher) and Agilent High Sensitivity DNA kit (Agilent). Libraries were mixed together and prepared at a final concentration of 12pM and run on MiSeq Reagent Kit v3 (Illumina) according to the manufacturer’s specifications. Adaptors, primer sequences and detailed protocol temperatures can be found in Table S2 .
4
m6A-SAC-seq of K562 caRNA and RBFOX2-bound RNA
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m6A-SAC-seq was performed following the previously published procedure36 (link). Briefly, 50 ng of K562 caRNA and RBFOX2-bound RNA were fragmented with RNA Fragmentation Reagents (Thermo Fisher Scientific, AM8740) at 70 °C for 5 min, followed by end repair with T4 Polynucleotide Kinase (New England Biolabs, M0201) at 37 °C for 1 h. Next, spike-in mix was added, and ligation of RNA 3′ biotinylated adaptor was performed by using T4 RNA ligase 2, truncated KQ (New England Biolabs, M0373). Excess adaptors were digested with 5′ deadenylase (New England Biolabs) and RecJ (New England Biolabs). RNAs were enriched by Dynabead MyOn Streptavidin C1 beads (Thermo Fisher Scientific, 65001) following the manufacturer’s instructions, and labelled by Mjdim1 with allyl-SAM for two rounds at 50 °C for 1 h, followed by iodine labelling at room temperature for another 1 h. Reverse transcription was carried out with recombinant HIV reverse transcriptase (Worthington Biochemical, LS05006) at 37 °C for 2 h, and template RNAs were digested with RNase H (New England Biolabs). The libraries were prepared by complementary DNA adaptor ligation with T4 RNA ligase 1, high concentration (New England Biolabs) and PCR amplification with NEBNext Multiplex Oligos for Illumina (New England Biolabs).
5
Small RNA Library Construction and Sequencing
6
Small RNA Demethylation and Sequencing
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Small RNA (<200 nt) was extracted and purified using miRNeasy Mini Kit (Qiagen) and RNeasy MinElute Cleanup Kit (Qiagen). Purified small RNA fragments were demethylated by wild-type and mutant AlkB, purified by phenol/chloroform extraction and ethanol precipitation, labeled by CMC. The demethylation reaction and CMC labeling were performed as described. 14 (link) Briefly, 50 ng small RNA was denatured at 65°C for 5 minutes (min) and demethylated by wild-type and D135S mutant AlkB. The purified small RNA was denatured at 80°C for 5 min, added to BEU buffer with or without CMC, incubated at 37°C for 20 min, then purified by ethanol precipitation. The purified RNA was dissolved in sodium carbonate buffer and shaken at 37°C for 6 hours. The library was established as described. 14, (link)36 (link) Briefly, small RNA was dephosphorylated with CIP (NEB). The 3' adaptor ligation was added with T4 RNA ligase2, truncated KQ (NEB), followed by 5' Deadenylase (NEB) and RecJf (NEB) digestion. RNA was reverse transcribed by SuperScript III reverse transcriptase (Invitrogen), then digested by RNase H. The 5' adaptor ligation was added with T4 RNA ligase 1, high concentration (NEB). The ligated cDNA was amplified by Q5 High-Fidelity 2× Master Mix (NEB). The purified libraries were sequenced on Illumina NovaSeq 6000.
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