A GBS strategy was used to develop SNP markers as previously described (Zhang et al., 2016 (link)). Briefly, approximately 0.1 to 1 µg of genomic DNA was incubated at 37°C with MseI (New England Biolabs, NEB), T4 DNA ligase (New England Biolabs, NEB), ATP (New England Biolabs, NEB), and an MseI Y adapter N containing barcodes, and the samples were then heat-inactivated at 65°C. Two additional enzymes, HaeIII and EcoRI (New England Biolabs, NEB), were simultaneously added into the MseI digestions to further digest the fragments at 37°C. Then, the digested fragments with ligations were purified with Agencourt AMPure XP beads (Beckman Coulter, Inc.) and subjected to PCR amplification with the Phusion Master Mix (New England Biolabs, NEB) using both universal primers as well as i5 and i7 index primers (Illumina). The PCR products were purified using Agencourt AMPure XP beads (Beckman Coulter, Inc.), pooled, and separated by electrophoresis on a 2% agarose gel. Fragments of 400 to 450 bp (with indexes and adaptors) were excised from the gel and purified using a gel extraction kit (QIAGEN). These purified products were further cleaned using Agencourt AMPure XP beads (Beckman Coulter, Inc.) prior to sequencing. Then, paired-end 150 bp sequencing was performed on the selected tags on the Illumina HiSeq 4000 platform.
Adenosine triphosphate (atp)
Manufactured by New England Biolabs
Sourced in United States
ATP (Adenosine Triphosphate) is a high-energy nucleotide compound that serves as the primary energy currency in living cells. It is involved in numerous cellular processes, including energy production, cell signaling, and metabolic regulation.
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Lab products found in correlation
T4 dna ligase, by New England Biolabs (21 mentions)
Gel extraction kit, by Qiagen (6 mentions)
Peg8000, by New England Biolabs (5 mentions)
Phusion master mix, by New England Biolabs (4 mentions)
Ecori, by New England Biolabs (4 mentions)
Cutsmart buffer, by New England Biolabs (4 mentions)
Splintr ligase, by New England Biolabs (4 mentions)
T4 rna ligase 1, by New England Biolabs (4 mentions)
E coli poly a polymerase, by New England Biolabs (4 mentions)
T4 polynucleotide kinase, by New England Biolabs (4 mentions)
T4 pnk, by New England Biolabs (4 mentions)
Agencourt ampure xp, by Beckman Coulter (3 mentions)
T4 rna ligase buffer, by New England Biolabs (3 mentions)
T4 dna ligase buffer, by New England Biolabs (3 mentions)
Gaiix, by Illumina (2 mentions)
Bst 2.0 dna polymerase, by New England Biolabs (2 mentions)
T4 dna ligase, by Thermo Fisher Scientific (2 mentions)
Small rna kit, by Agilent Technologies (2 mentions)
Zymo oligo clean concentrator kit, by Zymo Research (2 mentions)
Hiscribe t7 high yield rna synthesis kit, by New England Biolabs (2 mentions)
64 protocols using adenosine triphosphate (atp)
1
GBS Marker Development Protocol
2
RIG-I:RNA Complex Formation and Trypsin Digestion Assay
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Confluent 10-cm-diameter dishes of Huh7 or doxycycline-induced 293TdoxRIG-I cells were scraped in binding buffer (25 mM Tris HCl [pH 7.5], 150 mM NaCl, 1.5 mM MgCl2) supplemented with 0.5% Triton X-100 without protease inhibitors. The concentration of total protein in clarified lysates was determined by Bradford assay. The reaction volume was 500 µl and included 1 mg to 2 mg total lysate protein, RNA (33 nM to 1 µM), and 2 mM AMP-PNP (Roche) or ATP (New England Biolabs). The reaction mixtures were incubated for 30 min at room temperature to permit RIG-I:RNA complex formation. Next, tosylsulfonyl phenylalanyl chloromethyl ketone (TPCK)-treated trypsin (Sigma-Aldrich) was added to each reaction for a final mass ratio of 1:400, and the reaction mixture was incubated at room temperature. At the indicated time points (0 to 360 min), 25-µl aliquots were removed for boiling in SDS sample buffer. Experiments using multiple RNA concentrations were set up by serially diluting the RNA in binding buffer, with digestions performed for 1.5 h with a 1:200 mass ratio of trypsin. RIG-I fragments were visualized by immunoblotting using clone Alme-1 (Adipogen) mouse anti-RIG-I helicase antibody or monoclonal mouse anti-RIG-I CARDs (KeraFast).
3
In Vitro Synthesis of Capped Rab5ab RNA
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Capped rab5ab RNA was synthesised in vitro using 5 µg (5 µl) of linearized rab5ab DNA (IRAKp961M19104 sub-cloned into a pCS2+ vector or a GFP-pCS2+vector) in a 50 µl reaction containing 10 µl of 5× transcription buffer, 5 µl of 0.1 M DTT, 5 µl of 5 mM CAP (NEB), 5 µl of 1 mM GTP (NEB), 5 µl of 5 mM UTP (NEB), 5 µl of 5 mM ATP (NEB), 5 µl of 5 mM CTP (NEB), 2 µl of RNAse inhibitor (NEB) and 3 µl of SP6 RNA polymerase (NEB) incubated at 37 °C for 20 min when 4 µl of 10 mM GTP was added and incubated at 37 °C for a further 2 h. An additional 3 µl of RNAse free DNAse (Promega) was added and the reaction incubated at 37 °C for a further 20 min. The RNA was separated from the other reaction components using Chroma-100 spin columns (Clontech).
4
TALER RVD Sequence Generation
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All plasmid sequences were submitted to GenBank, with accession number KM486811 ~ KM486934 and KM519784 ~ KM519788.
TALER RVD sequences are listed inSupplementary Table 1 . When required, equal molar amounts of oligonucleotides were annealed in 1× PNK buffer by heating to 95 °C and gradually cooling down (− 1 °C per min) to 37 °C, and then 1 μM of annealed product was phosphorylated by 0.5 unit/μL PNK in presence of 0.5 mM ATP (New England Biolabs).
The vectors for making stable cell line inSupplementary Fig. 2d were made as described35 (link). Briefly, plasmid DNAs were pooled 7 fmol each and digested with I-SceI. The digested DNA mixture was added to a Gibson reaction buffer with integrative carrier vector and adapter vector.
TALER RVD sequences are listed in
The vectors for making stable cell line in
5
Purification and Phosphorylation of PKR
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In total, 1 × 107 Hela cells were lysed with 1 × RIP buffer and centrifuged at 13,000 rpm for 15 min at 4 °C. PKR was purified from the supernatants using protein A + G magnetic beads conjugated anti-PKR antibody: washed four times with 5 × RIP buffer, once with 1 × RIP buffer, and then resuspended in 1 × RIP buffer. Purified PACT and in vitro transcribed RNA were incubated with beads for 2 h at 4 °C, then washed three times with 1 × RIP buffer, once with kinase buffer (20 mM HEPES at pH 7.4, 40 mM MgCl2, 100 mM KCl, 1 mM DTT, PMSF, 0.5% NP-40, protease inhibitor cocktail, RNasin ribonuclease inhibitor, phosphatase inhibitor cocktail (Beyotime Biotechnology)), then resuspended in kinase buffer containing 50 μM ATP (NEB). After 30 min at 30 °C the reaction was terminated through a wash with 1 × RIP buffer. Proteins were eluted by 1 × SDS buffer and resolved on SDS-PAGE gel. Phospho-PKR were detected by WB.
6
Genotyping-by-sequencing for grassPea
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GBS sequencing was conducted according to the method of Elshire et al., 2011 [29 (link)], with minor improvements. Briefly, the gDNA from 154 grasspea plants (2 parents and 152 F2 progeny) was first digested by MseI (5’-T!TAA-3’) (New England Biolabs, ‘NEB’, Ipswich, MA, USA) at 37 °C, then subjected to restriction-ligation by T4 DNA ligase (NEB) and ATP (NEB) with a MseI Y-adapter N-containing barcode at 65 °C, followed by a second digestion with HaeIII (5’-GG!CC-3’) (NEB) at 37 °C. The digested fragments were purified using Agencourt AMPure XP (Beckman Coulter, ‘Beckman’, Brea, CA, USA) and used for PCR amplification, utilizing Phusion Master Mix (NEB) to add universal and index primers, as well as i5 and i7 index sequences, to the digested fragments. The purified PCR fragments (425–490 bp including indexes and adaptors) were screened and extracted using a 2% agarose gel with the Gel Extraction Kit (Qiagen, Valencia, CA, USA). The amplification products were purified using Agencourt AMPure XP (Beckman) and diluted before sequencing. Finally, an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) was used to perform 150-bp paired-end sequencing by Tianjin Novogene Technology Co., Ltd. (Tianjin, China).
7
Bulked Sequencing of Plant Genotypes
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Together with the parental lines, G21 and Jiliang 2, the bulked pools were sequenced on an Illumina GAIIx machine (Illumina, San Diego, CA, USA) according to Biomark’s instruction [26 (link)]. Briefly, genomic DNA from the parental lines and both bulked pools were incubated at 37°C with 0.6U MseI (New England Biolabs, Hitchin, Herts, UK), T4 DNA ligase (NEB), ATP (NEB) and MseI adapters. Restriction-ligation reactions were heat-inactivated at 65°C and then digested in an additional restriction with enzymes HaeIII and BfaI at 37°C. Then PCR was performed containing the diluted restriction-ligation samples, dNTP, Taq DNA polymerase (NEB) and MseI-primer containing a unique barcode for each sample. The PCR products were purified by using E.Z.N.A.H Cycle Pure Kit (Omega) and pooled.
8
Genotyping-by-Sequencing Library Preparation
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GBS libraries were constructed in accordance with the modified protocol [45 (link)]. Genomic DNA was incubated at 37℃ with MspI (New England Biolabs, NEB), T4 DNA ligase (NEB), ATP (NEB), and MspI Y adapter N-containing barcode. Restriction-ligation reactions were heat-inactivated at 65℃ and digested with the additional restriction enzyme EcoRI (NEB) at 37℃. The restricted ligation samples were purified using Agencourt AMPure XP (Beckman). The purified samples were PCR amplified with Phusion Master Mix (NEB) universal primer and index primer to add the index. The PCR products were purified, pooled, and electrophoresed on 2% agarose gel. Fragments between 265 and 315 bps were selected and purified. Pair-end sequencing was performed on the selected tags using the Illumina PE150 platform.
9
SELECT Analysis of METTL16 Targets
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SELECT analysis was performed as described previously60 (link). Briefly, total RNA was isolated using TRIzol followed by RNeasy Mini Kit (QIAGEN) with RNase-Free DNase Set (QIAGEN). Then an equal amount of RNA samples was mixed with 40 nM up and down probes and 5 μM dNTP in 1× CutSmart Buffer (New England Biolabs). Samples were denatured at 90 °C for 1 min, and then gradually cooled to 40 °C. Next, 0.01 U Bst 2.0 polymerase (New England Biolabs), 0.5 U SplintR ligase (New England Biolabs), and 10 nM ATP (New England Biolabs) were added to the reaction mixture. Samples were incubated at 40 °C for 20 min, then at 80 °C for 20 min, and cooled to 4 °C. The final reaction mixtures were used for qPCR analysis as described above. Primers used for SELECT analysis are listed in Supplementary Table 3 . METTL16 target and nontarget sites were chosen based on the MeRIP-seq results.
10
Targeted Detection of m6A Modifications
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Detection of m6A at targeted sites was based on the SELECT technology modified from a previous protocol (39 (link)). For each sample, 1 μg of total RNA was incubated with 40 nM Up primer, 40 nM Down primer and 5 μM dNTP (New England Biolabs, #N0446S) in 17.5 μl 1× CutSmart buffer (New England Biolabs, #B7204S). A progressive annealing cycle was carried out: 1 min each at 90°C, 80°C, 70°C, 60°C, 50°C and 40°C for 6 min. Subsequently, 2.5 μl of enzyme mixture containing 0.01 U Bst 2.0 DNA polymerase (New England Biolabs, #M0537S), 0.5 U SplintR ligase (New England Biolabs, #M0375S) and 10 nmol ATP (New England Biolabs, # P0756S) were added to the 17.5 μl annealing products. The final 20 μl reaction mixtures were incubated at 40°C for 20 min, denatured at 80°C for 20 min and then kept at 4°C. Afterward, 2 μl of final products were transferred to a reaction mixture containing 200 nM SELECT common primers and 2× SYBR Green Master Mix for qPCR analysis. The run cycle was set up as: 95°C for 1 min followed by 40 cycles of (95°C, 20 s; 60°C, 60 s). The SELECT products of targeted sites were normalized to the RNA abundance of corresponding transcripts containing m6A sites. All assays were performed with three independent experiments. Primers used in the SELECT assays were listed in Supplementary Table S4 .
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