Evaluation of RNA extraction and rRNA depletion protocols for RNA-Seq in eleven edible seaweed species from brown, red, and green algae.

Seaweeds represent a promising resource for food as well as pharmaceutical and cosmetic applications. However, genomic and transcriptomic research on these organisms remains underdeveloped, partly due to technical challenges in DNA/RNA extraction and ribosomal RNA (rRNA) depletion. These challenges are particularly acute in species lacking standardized protocols. This study systematically evaluates RNA isolation and rRNA depletion protocols for RNA sequencing (RNA-seq) across 11 commercially relevant edible seaweed species, encompassing six brown (Heterokontophyta), four red (Rhodophyta), and one green (Chlorophyta) alga. Seven RNA extraction protocols (three CTAB-based and four spin-column-based) were compared for RNA yield, integrity, and purity. Brown seaweeds generally yielded superior RNA with CTAB-based methods, whereas red and green seaweeds performed better with chaotropic-salt-based spin-column methods. Additionally, three commercial rRNA depletion kits (Ribo-Zero Plant, riboPOOL, and RiboFree) were assessed for each seaweed species separately in a large matrix-style RNA-seq experiment (43 samples) to determine their effectiveness in removing rRNA compared to undepleted controls. Both RiboFree and riboPOOL significantly outperformed Ribo-Zero Plant, yielding post-depletion rRNA mapping rates of only 6, 9, and 19%, respectively. This study provides practical guidelines for selecting RNA isolation and ribodepletion methods tailored to specific seaweed taxa and supports the development of transcriptomic tools for seaweed research.