Plant rna isolation aid reagent

The CRN and CRN/PPN1 strains were grown in control YPD and YPD with 5 mM of MnSO₄. After cultivation for 12 h (control, both strains), 40 h (manganese, CRN) and 16 h (manganese, CRN/PPN1), the cells were harvested by centrifugation at 5000× g for 10 min and washed three times with distilled water at 0 °C, centrifuged after each washing, and placed in RNALater. Two replicates were obtained from two simultaneous cultivations in control YPD and manganese-containing YPD. Total RNA extraction was performed using an RNeasy Plant Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s protocol—with the addition of Plant RNA Isolation aid reagent (Ambion/ThermoFisher Scientific, Waltham, MA, USA) to lysis buffer RLT. Illumina cDNA libraries were constructed using the TruSeq RNA Sample Prep Kits v2 (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol. The libraries were sequenced on the Illumina NextSeq 500 (Illumina, San Diego, CA, USA). The total sequenced library size was no less than 3 million reads per replicate. The raw and processed RNA-Seq data are deposited in GEO under accession number GSE122987.

The procedures for sample collection, DNA and RNA library preparation for most datasets (DNA shotgun and mate-pair libraries and an inflorescence transcriptome library) and sequencing were described in the reports of Logacheva et al. (2016) (link) and Schelkunov, Penin & Logacheva (2018) (link). In addition to the data generated in previous studies, we used four new datasets that represent the transcriptomes of the anthers and ovules of H. monotropa. The samples were collected in the same location as previous samples but in 2018. H. monotropa is not an endangered or threatened plant species; thus, no specific permissions were required for its collection. RNA was extracted using an RNEasy kit (Qiagen, Hilden, Germany) with the addition of the Plant RNA Isolation Aid reagent (Thermo Fisher, Waltham, MA, USA) to the lysis buffer. The removal of ribosomal RNA was performed using a Ribo-Zero plant leaf kit (Illumina, San Diego, CA, USA), and further sample preparation was performed using a NEBNext RNA library preparation kit (New England Biolabs, Ipswich, MA, USA). The libraries were sequenced on the HiSeq 4000 platform (Illumina, San Diego, CA, USA) in 150-nt paired-end mode at the Skoltech Genomics Core Facility. The reads were deposited in the NCBI Sequence Read Archive under BioProject PRJNA573526.