RNA extraction and RNA-Seq analysis
Cultures of S. meliloti , grown overnight in TY medium at 30°C at
130 rpm, were diluted to an OD600 0.1 in 5ml of TY
medium supplemented with 50% v/v of Trichoderma 1 week-spent
medium and incubated for 24 hours. Three biological replicates
(independent cultures) were produced for each strain-spent medium
combination, including untreated control with fresh medium. A total of
60 samples then were prepared (4 strains x 5 conditions x 3 replicas).
After incubation, cells were blocked with RNAprotect Bacteria (Qiagen,
Venlo, The Netherlands), and total RNA was extracted using RNeasy Mini
kits (Qiagen) from 0.5 ml of culture following the manufacturer’s
instructions, including on column DNase I treatment as reported in . In
particular, after elution, a second DNase I (ThermoFisher, Waltham,
Massachusetts, USA) treatment was performed, and the absence of
contaminant DNA was verified by qPCR on the nodC gene of S.
meliloti . Quality and quantity of total RNA were checked by Agilent
2200 TapeStation (Agilent) with RNA Screen Tape. NEBNext rRNA Depletion-
Bacteria (New England Biolabs) was used for ribosomal RNA depletion. The
library was prepared with TruSeq Stranded Total RNA Library Prep Gold
Kit (Illumina). Sequencing was performed on an Illumina Novaseq6000
apparatus by Macrogen Crop. (Korea)