*2.4. Transcriptome and Small RNA Sequencing*

Total RNA was extracted from 100 mg of tuber tissue partitioned from the sample taken for metabolite analysis using the RNeasy plant mini kit (Qiagen, Hilden, Germany). RNA quality and concentration were verified using an Agilent Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). TruSeq RNA and small RNA sequencing libraries were constructed following the standard preparation guide (Illumina, San Diego, CA, USA). All eight RNA samples (four replicates of each treatment) were multiplexed in a lane of a flow cell and paired-end sequencing (125 cycles) was performed using an Illumina HiSeq 2500. Similarly, for small RNA sequencing, all 8 samples were multiplexed in a lane of a flow cell and single-end sequencing was carried out on Illumina HiSeq 2500.

### *2.5. RNA and Small RNA Read Mapping and Analysis*

Before read mapping and expression quantification, all RNA reads were filtered using Trimmomatic (version 0.36; [22]) by (i) removing adapter sequences, (ii) trimming leading and trailing low-quality sequences, (iii) removing sequences when the average quality per base dropped below 15 within a 4-base wide sliding window and (iv) keeping only those pairs where both reads were longer than 75 bp. Clean reads were aligned to the potato reference genome (SolTub\_3.0, EnsemblPlants) with STAR (v2.5.2b) and isoform expression was quantified with the RSEM (v1.3.3) algorithm [23]. The expected read counts generated by the RSEM algorithm were rounded o ff and fed into DESeq2.

The quality of small RNA sequencing reads was assessed using the FASTQC program (v0.11.8; [24]). Reads were quality-filtered and adapter-trimmed using cutadapt (v2.8; [25]). The alignment of filtered reads to the potato reference genome (SolTub\_3.0, EnsemblPlants) and annotation and quantification of small RNAs was carried out using ShortStack (v3.8.5; [26]). psRNATarget [27] was used to predict the miRNA and small RNA target genes.
