Purelink plant rna reagent

RNAs were extracted from all inoculated and control plants 10 days after inoculation using PureLink™ Plant RNA Reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer‘s instructions. The total RNA yield and quality were measured using a Bioanalyzer 2100 (Agilent Technologies, Palo Alto, CA, USA) using the Agilent RNA 6000 Nano Kit and Modulus™ Single Tube Multimode Reader (Turner Biosystems, Sunnyvale, CA, USA) using the Quant-iT™ RNA Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). Samples with an RNA Integrity Number (RIN) below 7 were excluded from further analysis. Only RNA concentrations higher than 5 ng μL⁻¹ were used, and all samples at higher concentrations were diluted to 5 ng μL⁻¹ based on fluorimetry. After RNA quantification, samples were pooled in groups according to the variant of inoculation, resulting in a total of five replicates per variant.

Four replicates were sampled at each time point (d0, d1, d7, d8 and d14) from the W-B and D treatments for total of 36 samples. Total RNA was extracted from each cell suspension taking 0.1 mL approximately of pellet volume using combined protocols of PureLink Plant RNA Reagent (Ref: 12322012, Thermo Fisher Scientific) and RNeasy Plus Universal Mini Kit (Cat No. 73404, QIAGEN) following the manufacturer’s protocol. RNA concentrations and quality were measured using an Agilent 2100 bioanalyzer and a minimal RIN number of 7 was used for sequencing. The construction of the cDNA libraries and RNAseq were performed by the Genomics Core Facility at Penn State University (University Park, USA). cDNA libraries were prepared with a TruSeq stranded mRNA library prep Kit (cat# RS-122-2101, Illumina, San Diego, CA, USA). The libraries were sequenced on a HiSeq™ 2500 (Illumina) using single-end runs of 100 nt.

To annotate microRNAs (miRNAs) present in the Streptochaeta genome, we (i) sequenced sRNAs from leaf, anther and pistil tissues, (ii) compared miRNAs present in anthers to those of three other representative monocots (rice, maize, and asparagus), and (iii) validated gene targets of these miRNAs.
We collected tissues from leaf and pistil as well as 1.5, 3, and 4 mm anthers. Samples were immediately frozen in liquid nitrogen and kept at –80°C prior to RNA isolation. Total RNA was isolated using the PureLink Plant RNA Reagent (Thermo Fisher Scientific, Waltham, MA, United States). sRNA libraries were published previously (Patel et al., 2021 (link)). RNA sequencing libraries were prepared from the same material using the Illumina TruSeq stranded RNA-seq preparation kit (Illumina Inc., United States) following manufacturer’s instructions. Parallel analysis of RNA ends (PARE) libraries were prepared from a total of 20 μg of total RNA following the method described by Zhai et al. (2014) (link). For all types of libraries, single-end sequencing was performed on an Illumina HiSeq 2000 instrument (Illumina Inc., United States) at the University of Delaware DNA Sequencing and Genotyping Center.