Key resources table.
| Reagent type (species) or resource | Designation | Source orreference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Arabidopsis thaliana) | ECT2 | TAIR10 | AT3G13460 |
EVOLUTIONARILY CONSERVED C-TERMINAL REGION 2 |
| Gene (Arabidopsis thaliana) | ECT3 | TAIR10 | AT5G61020 |
EVOLUTIONARILY CONSERVED C- TERMINAL REGION 3 |
| Gene (Arabidopsis thaliana) | ECT4 | TAIR10 | AT1G55500 |
EVOLUTIONARILY CONSERVED C- TERMINAL REGION 4 |
| Gene (Drosophila melanogaster) | ADAR Isoform N | Genebank, FlyBase, NCBI |
CG12598 NM_001297862 | Adenosine deaminase acting on RNA |
| Strain (Escherichia coli) | DH5α | NEB | Cat. # 18258012 | MAX Efficiency DH5α Competent Cells |
| Strain (Agrobacterium tumefaciens) | GV3101 | Koncz and Schell, 1986 | ||
| Genetic reagent (A. thaliana) | SALK_002225 C (ect2-1) | NASC | N657472 N2110120 | |
| Genetic reagent (A. thaliana) | te234 (ect2-1/ect3-1/ect4-2) | Arribas-Hernández et al., 2018 | N2110132 | Donated to NASC and ABRC |
| Genetic reagent (A. thaliana) | ECT2pro:FLAG-DmADAR E488Q cd -ECT2ter | This paper (see Methods) |
Seed requests to pbrodersen@bio.ku.dk |
|
| Genetic reagent (A. thaliana) | ect2-1/ECT2pro:ECT2-FLAG-DmADAR E488Q cd -ECT2ter | This paper (see Methods) |
Seed requests to pbrodersen@bio.ku.dk |
|
| Genetic reagent (A. thaliana) | te234/ECT2pro:ECT2-FLAG-DmADAR E488Q cd -ECT2ter | This paper (see Methods) | Seed requests to pbrodersen@bio.ku.dk |
|
| Genetic reagent (A. thaliana) |
ect2-1/ECT2pro: ECT2-mCherry-ECT2ter |
Arribas-Hernández et al., 2018; Arribas-Hernández et al., 2020 | N2110839 N2110840 | Donated to NASC and ABRC |
| Genetic reagent (A. thaliana) |
ect2-1/ECT2pro: ECT2 W464A - mCherry-ECT2ter |
Arribas-Hernández et al., 2018; Arribas-Hernández et al., 2020 | N2110841 N2110842 | Donated to NASC and ABRC |
| Genetic reagent (A. thaliana) |
ect2−1/ ECT2pro:3xHA- ECT2-ECT2ter |
This paper (see Methods) |
Seed requests to pbrodersen@bio.ku.dk |
|
| Genetic reagent (A. thaliana) | ect2−1/ ECT2pro:3xHA-ECT2 W464A -ECT2ter | This paper (see Methods) |
Seed requests to pbrodersen@bio.ku.dk |
|
| Genetic reagent (D. melanogaster) | Canton-S | Bloomington Drosophila Stock Center | BDSC:64,349 | Used to extract RNA and produce cDNA for cloning |
| Antibody | anti-FLAG (mouse monoclonal) | Sigma-Aldrich | A8592 | Used for WB (1:1000) |
| Antibody | anti-mCherry (rabbit polyclonal) | Abcam | ab183628 | Used for WB (1:1000) |
| Antibody | anti-HA (mouse monoclonal) | Abnova | 12CA5 | Used for WB (1:2000) |
| Antibody | RFP-Trap RFP Nanobody/VHH coupled to agarose (recombinant, monoclonal) | ChromoTek | Cat. # rta-20 | Used for IP (20 μL of beads for 4 g of tissue in 6 mL of buffer) |
| Antibody | Anti-HA Affinity Matrix from IgG1 3 F10 (rat, monoclonal) |
Roche | Cat. # 11815016001 | Used for IP (10 μL of beads for 500 mg of tissue in 750 μL of buffer) |
| Recombinant DNA reagent | pCAMBIA3300U (plasmid) | Nour-Eldin et al., 2006 | Used for cloning | |
| Commercial assay or kit | pGEM -T Easy (plasmid and cloning kit) |
Promega | Cat. # A1360 | Used for cloning |
| Commercial assay or kit | KAPAHiFi HotStart Uracil + Kit |
Roche | Cat. # 7959079001 | Used for cloning |
| Commercial assay or kit | AccuPrime Supermix I | Invitrogen | Cat. # 12342–010 | Used for iCLIP library preparation |
| Peptide, recombinant protein | Uracil-DNA Glycosylase (USER enzyme) |
NEB | Cat. # M5505L | Used for cloning |
| Peptide, recombinant protein | Turbo DNase | Ambion | Cat. # AM2238 | Used for CLIP |
| Peptide, recombinant protein | RNase I | Ambion | Cat. # AM2294 | Used for CLIP |
| Peptide, recombinant protein | T4 Polynucleotide Kinase (PNK) | ThermoFisher Scientific |
Cat. # EK0031 | Used for iCLIP library preparation |
| Peptide, recombinant protein | T4 RNA Ligase I, High Concentration |
NEB | Cat. # M0437M | Used for iCLIP library preparation |
| Peptide, recombinant protein | Proteinase K | Roche | Cat. # 3115887001 | Used for iCLIP library preparation |
| Peptide, recombinant protein | Superscript III Reverse Transcriptase |
Invitrogen | Cat. # 18080–093 | Used for iCLIP library preparation |
| Peptide, recombinant protein | CircLigase II ssDNA Ligase |
Epicentre | Lucigen Cat. # CL9021K | Used for iCLIP library preparation |
| Peptide, recombinant protein | BamHI (Fast Digest) |
ThermoFisher Scientific |
Cat. # FD0054 | Used for iCLIP library preparation |
| Chemical compound, drug | cOmplete protease inhibitor cocktail |
Roche | Cat. # 11697498001 | Used for CLIP |
| Chemical compound, drug | Protease inhibitor cocktail for plant cell extracts |
Sigma | Cat. # P9599 | Used for CLIP |
| Chemical compound, drug | Glufosinate-ammonium (PESTANAL) | Sigma | Cat. # 45520 77182-82-2 | Used for selection of transgenic lines |
| Sequence-based reagent | Pre-adenylated adapter for iCLIP (3’-RNA linker) |
Huppertz et al., 2014 | L3-App | rAppAGATCG GAAGAGCGGT TCAG/ddC/ |
| Sequence-based reagent | iCLIP RT-primers (Two-part cleavable DNA adapters complementary to the 3’ RNA linker) |
Huppertz et al., 2014 | Rt1clip-Rt12clip | Used for iCLIP library preparation (seq: ) |
| Sequence-based reagent | USER and site-directed mutagenesis primers |
This paper (Appendix) |
Used for cloning. Sequences are in the Appendix | |
| Sequence-based reagent | Primers for detection of point mutations |
This paper (Appendix) |
Used for cloning. Sequences are in the Appendix |
|
| Software, algorithm | R | https://www.R-project.org/ | Used for data analyses | |
| Software, algorithm | hyperTRIBER | Rennie et al., 2021; https://github.com/sarah-ku/hyperTRIBER; https://github.com/sarah-ku/targets_arabidopsis | Used for calling significant ADAR-edited sites. Contact: sarah@binf.ku.dk |
|
| Software, algorithm | trimmomatic | Bolger et al., 2014 | Used for trimming RNAseq-reads | |
| Software, algorithm | STAR | Dobin et al., 2013 | Used for mapping RNAseq-reads | |
| Software, algorithm | Salmon | Patro et al., 2017 | Used for transcript quantification | |
| Software, algorithm | SAMtools mpileup | Li et al., 2009 | Used to count nt-mismatches | |
| Software, algorithm | rtracklayer | Lawrence et al., 2009 | Used to retrieve sequences | |
| Software, algorithm | ggseqlogo | Wagih, 2017 | Used to generate motif logos |
|
| Software, algorithm | Hmisc | https://github.com/harrelfe/Hmisc/ | Used for expression-based binning | |
| Software, algorithm | fastqc | https://www.bioinformatics.babraham.ac.uk/projects/fastqc/ | Used for quality control | |
| Software, algorithm | cutadapt | Martin, 2011 | Used for trimming of iCLIP reads | |
| Software, algorithm | flexbar | Roehr et al., 2017 | Used for demultiplexing iCLIP reads | |
| Software, algorithm | PureCLIP | Krakau et al., 2017 | Used for calling iCLIP peaks | |
| Software, algorithm | GenomicRanges | Lawrence et al., 2013 | Used to retrieve short sequences | |
| Software, algorithm | ‘Distributions of motifs per 1,000 sites over distance’ |
This paper https://github.com/sarah-ku/targets_arabidopsis |
Used to calculate motif distributions around m6A/iCLIP. Contact: sarah@binf.ku.dk | |
| Software, algorithm | ggplot2 | https://ggplot2.tidyverse.org | Used to generate plots | |
| Software, algorithm | bedtools | Dale et al., 2011; Quinlan and Hall, 2010 | Used to filter and clean iCLIP data | |
| Software, algorithm | Homer | Heinz et al., 2010 | Used for de novo motif discovery |
|
| Software, algorithm | FIMO | Grant et al., 2011 | Used to detect motif occurrences |
|
| Software, algorithm | gbm | https://github.com/gbm-developers/gbm | Used for random forest analysis |
|
| Software, algorithm | pROC | Robin et al., 2011 | Used to estimate predictive score of RF | |
| Software, algorithm | IGV (Integrative Genomics Viewer) | Robinson et al., 2011 | Used to show genomic data |