title: GSM7116233: Lobaria pulmonaria thallus, 11th day of experiment, replicate 2; Lobaria pulmonaria; RNA-Seq
library_construction_protocol: Samples for RNA extraction were collected after the UV treatment of lichen thalli, immediately fixed in liquid nitrogen and stored at -80 °C until further use. Extraction of RNA was performed using the RNeasy Plant Mini Kit (QIAGEN) with an on-column DNase digestion step (DNase 1, QIAGEN) according to the manufacturer's protocol. The quality of the extracted RNA was assessed using a Bioanalyzer 2100 (Agilent Technologies, USA). RNA concentrations were measured using a NanoDrop–1000 (Thermo Scientific, USA) and a Qubit fluorimeter (Life Technologies, Carlsbad, USA) with the RNA HS assay kit (QIAGEN). The NEBNext® Poly(A)+ mRNA isolation module (NEB, USA) with magnetic particles was used to isolate the mRNA fraction from 1 μg total RNA. cDNA libraries were prepared using a NEBNext® Ultra™ II Directional Kit (NEB, USA). PCR enrichment was carried out as follows: 98 °C for 30 s for initial denaturation followed by eight cycles of 98 °C for 10 s and 65 °C for 75 s with a final elongation 65 °C for 4 min. The quality of cDNA libraries was monitored using a Bioanalyzer 2100 (Agilent Technologies, USA) and a Qubit fluorimeter (Life Technologies, Carlsbad, USA) with the DNA HS assay kit (QIAGEN). Sequencing of cDNA libraries was carried out on the HiSeq 2500 platform using a HiSeq SR Cluster Kit v4 cBot and a HiSeq SBS Kit V4 50 cycle kit with 50-bp single-ended reads.