Qbaseplus software v2

The gene expression analysis was carried out on biological triplicates using RT-qPCR. After grinding, RNA was extracted using the RNeasy Plant Mini Kit (Qiagen), treated with DNase I on column, and characterised with a NanoDrop 1000 Spectrophotometer (Thermo Scientific) and a 2100 Bioanalyzer (Agilent Life Sciences). The RNAs displayed a RIN value between 7.9 and 10. Reverse transcription was carried out with the ProtoScript II Reverse Transcriptase (NEB) following the manufacturer’s instructions. Primers were designed with Primer3 and validated for the absence of dimers and secondary structures (hairpin) using OligoAnalyzer 3.1 (http://eu.idtdna.com/calc/analyzer). qPCR runs were performed in 384 well-plates with the Takyon SYBR Green low ROX (Eurogentec), on a ViiA7 Real-Time PCR System (Applied Biosystems). The specificity of the products was checked at the end of each run with the melt curve. Relative gene expressions were determined using the qBase^PLUS software v2.5 (Biogazelle). CsaETIF3e and CsaETIF3h were the most stable reference genes among ETIF3e, ETIF3h, Tubulin and ETIF4e. The genes are named based on the putative orthology with the genes from Arabidopsis.

The RNA-Seq data were validated with RT-qPCR using a subset of 10 genes involved in cell wall biogenesis. Reverse transcription was carried out with the ProtoScript II Reverse Transcriptase (NEB) following the manufacturer’s instructions. Primers were designed with Primer3 and validated for the absence of dimers and secondary structures (hairpin) using OligoAnalyzer 3.1². qPCR runs were performed in 384 well-plates, on a ViiA7 Real-Time PCR System (Applied Biosystems) with the Takyon SYBR Green low ROX (Eurogentec). A melt curve was realized at the end of each experiment to ensure the specificity of the products. Relative gene expressions were determined with the qBase^PLUS software v2.5 (Biogazelle). The geNorm analysis (Vandesompele et al., 2002 ) designated CsaETIF4E and CsaGAPDH as the most suitable genes for normalization (among Histone, EF2, Actin, Cyclophilin, Ubiquitin, GAPDH, Tubulin, ETIF4E, ETIF3H, and ETIF3E). Calibrated Normalised Relative Quantities (CNRQ) from RT-qPCR were used for the final comparison with the RPKM values.