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This revolutionary method represents a considerable breakthrough for non-model species since it allows enhanced gene annotation and gene expression studies when compared to former sequencing methods. However, woody plant tissues are challenging to the successful preparation of cDNA libraries, thus, impairing further cutting-edge sequencing analyses. Here, a detailed protocol for preparing cDNA libraries suitable for high throughput RNA sequencing using Oxford Nanopore Technologies\u00ae is described. This method was used to prepare eight barcoded cDNA libraries from two Solanum betaceum cell lines: one with compact morphology and embryogenic competency (EC) and another with friable and non-embryogenic (NEC). The libraries were successfully sequenced, and data quality assessment showed high mean quality scores. Using this method, long-read sequencing will allow a comprehensive analysis of plant transcriptomes.<\/jats:p>","DOI":"10.3390\/mps6020031","type":"journal-article","created":{"date-parts":[[2023,3,15]],"date-time":"2023-03-15T06:53:32Z","timestamp":1678863212000},"page":"31","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["An Efficient Method to Prepare Barcoded cDNA Libraries from Plant Callus for Long-Read Sequencing"],"prefix":"10.3390","volume":"6","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0147-0499","authenticated-orcid":false,"given":"Daniela","family":"Cordeiro","sequence":"first","affiliation":[{"name":"Centre for Functional Ecology, TERRA Associate Laboratory, Department of Life Sciences, University of Coimbra, Cal\u00e7ada Martim de Freitas, 3000-456 Coimbra, Portugal"}]},{"given":"Alexandra","family":"Camelo","sequence":"additional","affiliation":[{"name":"Centro de Apoio Tecnol\u00f3gico Agro-Alimentar (CATAA) de Castelo Branco, 6000-459 Castelo Branco, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3891-285X","authenticated-orcid":false,"given":"Ana Carolina","family":"Pedrosa","sequence":"additional","affiliation":[{"name":"Centre for Functional Ecology, TERRA Associate Laboratory, Department of Life Sciences, University of Coimbra, Cal\u00e7ada Martim de Freitas, 3000-456 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3767-9515","authenticated-orcid":false,"given":"In\u00eas","family":"Brand\u00e3o","sequence":"additional","affiliation":[{"name":"Centre for Functional Ecology, TERRA Associate Laboratory, Department of Life Sciences, University of Coimbra, Cal\u00e7ada Martim de Freitas, 3000-456 Coimbra, Portugal"},{"name":"Centro de Apoio Tecnol\u00f3gico Agro-Alimentar (CATAA) de Castelo Branco, 6000-459 Castelo Branco, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2299-298X","authenticated-orcid":false,"given":"Jorge","family":"Canhoto","sequence":"additional","affiliation":[{"name":"Centre for Functional Ecology, TERRA Associate Laboratory, Department of Life Sciences, University of Coimbra, Cal\u00e7ada Martim de Freitas, 3000-456 Coimbra, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9800-4186","authenticated-orcid":false,"given":"Christophe","family":"Esp\u00edrito Santo","sequence":"additional","affiliation":[{"name":"Centre for Functional Ecology, TERRA Associate Laboratory, Department of Life Sciences, University of Coimbra, Cal\u00e7ada Martim de Freitas, 3000-456 Coimbra, Portugal"},{"name":"Centro de Apoio Tecnol\u00f3gico Agro-Alimentar (CATAA) de Castelo Branco, 6000-459 Castelo Branco, Portugal"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2151-3916","authenticated-orcid":false,"given":"Sandra","family":"Correia","sequence":"additional","affiliation":[{"name":"Centre for Functional Ecology, TERRA Associate Laboratory, Department of Life Sciences, University of Coimbra, Cal\u00e7ada Martim de Freitas, 3000-456 Coimbra, Portugal"},{"name":"InnovPlantProtect CoLab, Estrada de Gil Vaz, 7350-478 Elvas, Portugal"}]}],"member":"1968","published-online":{"date-parts":[[2023,3,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"258","DOI":"10.1038\/ng.282","article-title":"A transcriptome atlas of rice cell types uncovers cellular, functional and developmental hierarchies","volume":"41","author":"Jiao","year":"2009","journal-title":"Nat. Genet."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Tian, C., Wang, Y., Yu, H., He, J., Wang, J., Shi, B., Du, Q., Provart, N.J., Meyerowitz, E.M., and Jiao, Y. (2019). A gene expression map of shoot domains reveals regulatory mechanisms. Nat. Commun., 10.","DOI":"10.1038\/s41467-018-08083-z"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Zhao, L., Zhang, H., Kohnen, M.V., Prasad, K.V.S.K., Gu, L., and Reddy, A.S.N. (2019). Analysis of transcriptome and epitranscriptome in plants using Pacbio iso-seq and Nanopore-based direct RNA sequencing. Front. Genet., 10.","DOI":"10.3389\/fgene.2019.00253"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"1348","DOI":"10.1038\/s41587-021-01108-x","article-title":"Nanopore sequencing technology, bioinformatics and applications","volume":"39","author":"Wang","year":"2021","journal-title":"Nat. Biotechnol."},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"892","DOI":"10.2144\/01304pf02","article-title":"Reverse transcriptase template switching: A SMARTTM approach for full-Length cDNA library construction","volume":"30","author":"Zhu","year":"2001","journal-title":"Biotechniques"},{"key":"ref_6","doi-asserted-by":"crossref","unstructured":"Marx, V. (2023). Method of the Year 2022: Long-read sequencing. Nat. Methods, 20.","DOI":"10.1038\/s41592-022-01759-x"},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Srivathsan, A., Lee, L., Katoh, K., Hartop, E., Kutty, S.N., Wong, J., Yeo, D., and Meier, R. (2021). ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone. BMC Biol., 19.","DOI":"10.1186\/s12915-021-01141-x"},{"key":"ref_8","doi-asserted-by":"crossref","unstructured":"Bayega, A., Oikonomopoulos, S., Wang, Y.C., and Ragoussis, J. (2022). Improved Nanopore full-length cDNA sequencing by PCR-suppression. Front. Genet., 13.","DOI":"10.3389\/fgene.2022.1031355"},{"key":"ref_9","first-page":"15","article-title":"Extraction of plant RNA","volume":"353","author":"MacRae","year":"2007","journal-title":"Methods Mol. Biol."},{"key":"ref_10","doi-asserted-by":"crossref","unstructured":"Vennapusa, A.R., Somayanda, I.M., Doherty, C.J., and Jagadish, S.V.K. (2020). A universal method for high-quality RNA extraction from plant tissues rich in starch, proteins and fiber. Sci. Rep., 10.","DOI":"10.1038\/s41598-020-73958-5"},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Rezadoost, M.H., Kordrostami, M., and Kumleh, H.H. (2016). An efficient protocol for isolation of inhibitor-free nucleic acids even from recalcitrant plants. 3 Biotech, 6.","DOI":"10.1007\/s13205-016-0375-0"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Zeng, Y., and Yang, T. (2012). RNA isolation from highly viscous samples rich in polyphenols and polysaccharides. Plan Mol. Biol. Rep., 20.","DOI":"10.1007\/BF02772130"},{"key":"ref_13","doi-asserted-by":"crossref","unstructured":"Inglis, P.W., Marilia de Castro, R.P., Resende, L.V., and Grattapaglia, D. (2018). Fast and inexpensive protocols for consistent extraction of high quality DNA and RNA from challenging plant and fungal samples for high-throughput SNP genotyping and sequencing applications. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0206085"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3159","DOI":"10.1105\/tpc.113.116053","article-title":"Plant callus: Mechanisms of induction and repression","volume":"25","author":"Ikeuchi","year":"2013","journal-title":"Plant Cell"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"323","DOI":"10.1007\/s11240-009-9531-0","article-title":"Differences in cell wall polysaccharide composition between embryogenic and non-embryogenic calli of Medicago arborea L.","volume":"97","author":"Endress","year":"2009","journal-title":"Plant Cell Tissue Organ Cult."},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Feh\u00e9r, A. (2019). Callus, dedifferentiation, totipotency, somatic embryogenesis: What these terms mean in the era of molecular plant biology?. Front. Plant Sci., 10.","DOI":"10.3389\/fpls.2019.00536"},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Correia, S., Alhinho, A.T., Casimiro, B., Miguel, C.M., Oliveira, M., Ver\u00edssimo, P., and Canhoto, J. (2019). NEP-TC a rRNA methyltransferase involved on somatic embryogenesis of tamarillo (Solanum betaceum Cav.). Front. Plant Sci., 10.","DOI":"10.3389\/fpls.2019.00438"},{"key":"ref_18","doi-asserted-by":"crossref","unstructured":"Jain, S.M., and Gupta, P.K. (2018). Step Wise Protocols for Somatic Embryogenesis of Important Woody Plants, Springer.","DOI":"10.1007\/978-3-319-79087-9"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"473","DOI":"10.1111\/j.1399-3054.1962.tb08052.x","article-title":"A revised medium for rapid growth and bio assays with tobacco tissue cultures","volume":"15","author":"Murashige","year":"1962","journal-title":"Physiol. Plant"},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Nath, O., Fletcher, S.J., Hayward, A., Shaw, L.M., Agarwal, R., Furtado, A., Henry, R.J., and Mitter, N. (2022). A comprehensive high-quality DNA and RNA extraction protocol for a range of cultivars and tissue types of the woody crop avocado. Plants, 11.","DOI":"10.3390\/plants11030242"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"77","DOI":"10.1111\/1755-0998.12938","article-title":"Harnessing the MinION: An example of how to establish long-read sequencing in a laboratory using challenging plant tissue from Eucalyptus pauciflora","volume":"19","author":"Schalamun","year":"2019","journal-title":"Mol. Ecol. Resour."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"186","DOI":"10.1101\/gr.8.3.186","article-title":"Base-calling of automated sequencer traces using Phred. II. Error probabilities","volume":"8","author":"Ewing","year":"1998","journal-title":"Genome Res."},{"key":"ref_23","doi-asserted-by":"crossref","unstructured":"Rivara-Espasand\u00edn, M., Balestrazzi, L., Dufort, G.A., Ochoa, I., Seroussi, G., Smircich, P., Sotelo-Silveira, J., Mart\u00edn, \u00c1., and Bateman, A. (2022). Nanopore quality score resolution can be reduced with little effect on downstream analysis. Bioinform. Adv., 2.","DOI":"10.1093\/bioadv\/vbac054"},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Delahaye, C., and Nicolas, J. (2021). Sequencing DNA with Nanopores: Troubles and biases. PLoS ONE, 16.","DOI":"10.1371\/journal.pone.0257521"}],"container-title":["Methods and Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2409-9279\/6\/2\/31\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,10]],"date-time":"2025-10-10T18:55:48Z","timestamp":1760122548000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2409-9279\/6\/2\/31"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2023,3,15]]},"references-count":24,"journal-issue":{"issue":"2","published-online":{"date-parts":[[2023,4]]}},"alternative-id":["mps6020031"],"URL":"https:\/\/doi.org\/10.3390\/mps6020031","relation":{"has-preprint":[{"id-type":"doi","id":"10.21203\/rs.3.rs-2097064\/v1","asserted-by":"object"}]},"ISSN":["2409-9279"],"issn-type":[{"value":"2409-9279","type":"electronic"}],"subject":[],"published":{"date-parts":[[2023,3,15]]}}}