title: GSM8644360: sample_10_Hardening_poly_rep2; Solanum lycopersicum; RNA-Seq
library_construction_protocol: Polysome profiling was performed according to [Lecampion et al., 2016 (https://doi.org/10.3791/54231)]. Briefly:  - 4-layer gradient (50%, 35% and 2 layers of 20% sucrose) - saline buffer (400 mM Tris-HCl pH 8.4; 200 mM KCl; 100 mM MgCl2) - polysome buffer (saline buffer 4x; EGTA 5.26 mM; Triton x100 0.5%; cycloheximide 50 μg/ml; chloramphenicol 50 μg/ml) - refrigerated centrifugation (4°C) for 2 hours 45 minutes at 175,000 g - precipitation with isopropanol  Total RNA from plants was isolated by phenol-chloroform extraction using ExtractRNA reagent (Eurogen, Russia, cat. no. BC032) according to the manufacturer's protocol. The obtained samples of total RNA were treated with DNaseI (Thermo scientific, USA, cat. no. EN0521) according to the manufacturer's protocol. Quantitative assessment and purification quality of the isolated RNA samples were determined by spectrophotometric method using a photometer for Synergy H1 microplates (BioTek, USA) and by electrophoresis in 1% agarose gel containing formaldehyde. TruSeq mRNA Stranded (Illumina) total RNA samples were enriched for the poly(A+) fraction, and then cDNA was synthesized from the stranded primer. The resulting cDNA was used to prepare libraries compatible with Illumina sequencing technology. The quality of the obtained libraries was checked using the Fragment Analyzer. Quantitative analysis was performed using qPCR. After quality control and DNA quantity assessment, the library pool was sequenced on an Illumina NovaSeq 6000 (read length - 150 bp on both sides of the fragments).
identifiers.namespace: GEO
identifiers.id: GSM8644360_r1