Agilent 2100 rna bioanalyzer

Frozen samples were first ground in a mill (M 20 Universal mill, Ika) previously cooled with liquid nitrogen and then transferred to a cooled mortar and reduced to a fine powder. Total RNA was isolated following a protocol described by Reid et al. with some minor modifications [139 ]. RNA isolation was performed separately by developmental stage, date of collection and mother tree.
Total RNA was purified using the RNeasy MinElute Cleanup kit (Qiagen) with on-column DNase I treatment (Qiagen RNase-Free DNase Set) and only samples with A_260/280 > 1.8 were used for further steps. RNA integrity was assessed in 1 % (w/v) agarose gels after ethidium bromide staining and for a rigorous assessment of RNA quality, the RNA samples were run on a RNA Pico6000 chip in Agilent 2100 Bioanalyzer RNA (Agilent). Additionally, each sample was quantified by fluorescence with the Quant-iT Ribogreen RNA Assay kit (Invitrogen).

‘TruSeq SmallRNA Sample Prep kit’ (Illumina, San Diego, CA) was used for library preparation following the manufacturer’s instructions. RNA samples were previously quantified, and quality tested by Agilent 2100 Bioanalyzer RNA (Agilent technologies, Santa Clara, CA) or by Caliper RNA LabChip GX (Caliper Life Sciences, Hopkinton, MA). Final libraries were quantified using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) and quality tested by Agilent 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA) or by Caliper RNA LabChip GX (Caliper Life Sciences, Hopkinton, MA). Libraries were then prepared for sequencing and sequenced on single-end 75 bp mode on NextSeq500 (Illumina, San Diego, CA). The Bcl2Fastq 2.0.2 version of the Illumina pipeline was used to process raw data for both format conversion and de-multiplexing. Adapter sequences were masked with Cutadapt v1.11 from raw Fastq data using the following parameters: -anywhere (on both adapter sequences) -overlap 5 -times 2 -minimum-length 35 -mask-adapter. Lower quality bases and adapters were trimmed by ERNE v1.4.6 software. Above mentioned small RNA-Seq procedures were commercially performed by IGA Technology Services (IGAtech), Udine, Italy (Fig. 1b). GLM analysis was performed cumulatively for all the four groups using DESeq software taking into consideration log2 fold change > ± 0.58 and p < 0.05^{55 (link)}.

Roots of control and treated seedlings were collected at 6 h and 15 d after treatment, immediately frozen in liquid N₂, and stored at −80 °C for further RNA-seq assays. Every RNA sample was derived from five independent seedlings. Total RNA was extracted from root samples using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) [56 ]. To eliminate genomic DNA contamination, the RNA samples were treated with RNase-free DNase I (Takara, Tokyo, Japan). The quality of the RNA was checked using an Agilent 2100 RNA Bioanalyzer (Agilent, Santa Clara, CA, USA). Libraries for RNA-Seq were prepared by using the VAHTS mRNA-Seq v2 Library Prep Kit (Vazyme Biotech Co., Ltd., Nanjing, China). Sequencing was performed using a HiSeqXTen sequencing system using stranded paired-end 150 bp sequencing (Illumina, Inc., San Diego, CA, USA).