RNA Extraction and Sequencing from Leaf Tissue

RNA was extracted from small pieces of stored samples (RNAlater; ThermoFisher) that were homogenized (PowerLyzer 24; Qiagen) and processed according to the standard protocol of the RNeasy PowerPlant Kit (Qiagen). We consistently excised the mid portion of the youngest fully mature leaf in the shoot (usually the second-rank leaf), so that tissue-age dependent within- or among-leaf variations were unlikely to explain differences in gene expression (Ruocco et al., 2019a (link),b (link)). RNA concentrations were measured with Qubit™ RNA HS Assays (ThermoFisher) using a Qubit 4 fluorometer. RNA integrity was verified on TapeStation 2200 with RNA ScreenTape. RIN values were >3.6.
RNA libraries were prepared from 1 μg of extracted RNA with NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina (New England Biolabs) following the polyA mRNA workflow of the kit protocol. Libraries were verified on TapeStation 2200 using High Sensitivity D1000 ScreenTape. Paired-end (2 × 150 bp) RNA sequencing (RNASeq) data were generated using a NextSeq 500/550 High Output Kit v2 (Illumina) on an Illumina NextSeq 500 platform at Nord University.

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Jueterbock A., Duarte B., Coyer J., Olsen J.L., Kopp M.E., Smolina I., Arnaud-Haond S., Hu Z.M, & Hoarau G. (2021). Adaptation of Temperate Seagrass to Arctic Light Relies on Seasonal Acclimatization of Carbon Capture and Metabolism. Frontiers in Plant Science, 12, 745855.

Publication 2021

RNAlater PowerLyzer 24 RNeasy PowerPlant Kit Qubit™ RNA HS Assays NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina NextSeq 500/550 High Output Kit v2 NextSeq 500 platform

Assays Gene expression Leaf Library Polya mrna Rna ii Sensitivity Tissue

Corresponding Organization :

Other organizations : Nord University, University of Lisbon, University of New Hampshire, University of Groningen, Marine Biodiversity Exploitation and Conservation, Qingdao National Laboratory for Marine Science and Technology, Peuplements végétaux et bioagresseurs en milieu tropical

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Gene expression

control variables

Tissue-age dependent within- or among-leaf variations were unlikely to explain differences in gene expression by consistently excising the mid portion of the youngest fully mature leaf in the shoot
RNA integrity was verified (RIN values were >3.6)

controls

No positive or negative controls were explicitly mentioned

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