title: GSM8170489: Cryopreserved, Dark, rep2; Solanum tuberosum; RNA-Seq
library_construction_protocol: For cryopreservation, shoot tips (2–3 mm) of 3.5-week-old in vitro plants were aseptically dissected under a stereomicroscope. The non-cryopreserved control shoot tips were trimmed to 1 mm. The explants were placed onto 4.5-cm sterile filter papers moistened with 2 ml of liquid MS medium for overnight. Cryopreservation was carried out using the DMSO-droplet method. Briefly, before cryopreservation, shoot tips were treated with cryoprotectant solution (10% DMSO (v/v)) in MS medium containing 30% sucrose for 2 h at room temperature (RT) before being transferred into droplets of 2.5 µl cryoprotectant solution on aluminium foil strips (0.5 x 1.5 cm). After that, the shoot tips were flash-frozen in liquid nitrogen (LN). The shoot tips were kept in LN for 1 h. After that, the shoot tips were rewarmed quickly by transferring the aluminium foil strips into liquid MS medium (50 ml) for 1.5 h at RT. After thawing, the cryopreserved samples (+) were placed on regeneration medium (MS with 0.5 mg/l zeatin riboside, 0.2 mg/l gibberellic acid (GA3), 0.5 mg/l indole acetic acid (IAA), and 30 g/l sucrose) on small Petri dishes (5.5 cm in diameter) and were allowed to regenerate for 48 h in total darkness. After the 48-h dark-incubation, the cryopreserved (+) samples were exposed to three different light conditions: total darkness (D), cool white fluorescent tubes (CW) and a combination of blue and red LEDs (90% red, 10% blue) (RB). The non-cryopreserved control samples (-) were only exposed to CW light. The light treatments lasted for 16 h, after which the samples (ten shoot tips per each treatment with three biological replicates) were collected into RNALater and stored at -80oC until RNA extraction. The intensity of both RB and CW was 40 µmol m-2s-1. Approximately 1 µg total RNA (RIN >7.4) was used for library preparation using Illumina's TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA) following the manufacturer's instructions. Quality of the libraries was assessed using an Agilent Bioanalyzer 2100 and concentrations with Qubit® Fluorometric Quantitation (Life Technologies). The obtained 12 libraries were pooled and run in one lane using an Illumina HiSeq2500 in high-output, paired-ended, 100 cycle mode, followed by read extraction (FastQ format) using bcl2fastq v1.8.4 (supported by Illumina).