4.2. RNA-Seq Experimental Design and RNA Extraction

The RNA-seq experimental design included six biological replicates each of the GR and GS biotypes, three with glyphosate treatment (2160 g ae. ha⁻¹) and three without treatment, giving a total of 12 plants. Three plants of each GR and GS biotypes were treated with glyphosate at 2160 g ae ha⁻¹, according to the method described above. At 24 h after treatment, the second and third leaves (from the apex) from all treated and non-treated plants were harvested and immediately frozen in liquid nitrogen and stored at −80 °C. Each plant formed an individual sample (Figure 6).

The RNA-seq experimental design using glyphosate-resistant (GR) and glyphosate-sensitive (GS) biotypes of Lolium multiflorum. RNA samples 1–3 and 4–6: GS and GR untreated, respectively; RNA samples 7–9 and 10–12: GS and GR, respectively, treated with glyphosate at 2160 g ae ha⁻¹ and harvested at 24 h after treatment. Three biological replicates were used per treatment (GS t0, GR t0, GS t1, and GR t1—a total of 12 libraries). Two biotypes originating from the same geographical region (São Valentin, RS, Brazil) were used, one GR and the other GS.

The Trizol reagent (Invitrogen, Carlsbad, Calif, USA) was used for the RNA extraction following the company’s recommendation. The residual genomic DNA was removed with DNase I (Invitrogen). The final experimental design comprised 12 RNA samples: 3 GS untreated (GS t0), 3 GR untreated (GR t0), 3 GS treated (GS t1), and 3 GR treated (GR t1) (Figure 6).