Transcriptome Profiling of Marchantia Liverworts

Gemmae of WT, Mpaccb8a-1/b-4, and Mpaccb8a-2/b-3 were incubated on half-strength Gamborg’s B5 medium for 2 weeks at 22 °C under continuous light. After 2 week-culture, the thalli were transferred to the medium with or without phosphate. Three biological replicates were prepared for each genotype. Total RNA was extracted using the NucleoSpin RNA Plant kit (Macherey-Nagel, Germany). mRNA was isolated from the total RNA with an NEBNext poly(A) mRNA Magnetic Isolation Module Kit (New England Biolabs, MA, USA). RNA-seq libraries were synthesized using an NEBNext Ultra RNA Library Prep Kit for Illumina and an NEBNext Adaptor for Illumina Kit (New England Biolabs, MA, USA). Paired-end sequencing was performed using the HiSeqXten (Illumina, CA, USA).
The reads were mapped onto the Marchantia paleacea genome^{3 (link)} assembly using HISAT2^{72 (link)} for Galaxy (https://usegalaxy.org) with default parameters. Read counts were calculated by Feature Counts for Galaxy. To identify differentially expressed genes, q values were calculated by the TCC R package^{73 (link)}, and genes with q value <0.05 and log2-fold change >1 or log2-fold change <−1 were selected as up-or down-regulated genes. Summary statistics of RNA-seq analysis are available in Supplementary Data 3.