GSM8173113: seedling roots, Po_GAQ1-treatment, 24 h, rep 3; Solanum lycopersicum; RNA-Seq
RNA was extracted using a Trizol-based method (Accurate Biology, China), and samples were stored at -80°C. The samples were ground in 2 mL tubes with liquid nitrogen, and 1 mL of TRIzol reagent (Accurate Biology, China) was added, mixed, and incubated for 5 min, and then 200 μL of chloroform was added, shaken, incubated for 5 min, and then centrifuged for 15 min at 12,000 x g at 4℃. The supernatant was transferred to a new tube, and an equal volume of pre-cooled isopropanol was added and mixed well, and then allowed to stand for 30 min at -80℃, and then centrifuged at 12,000 x g for 10 min at 4℃. The pellet was washed with 1 mL of 75% ethanol, and later dissolved in 30 μL of DEPC-treated water. RNA samples were analyzed by Nanodrop OD260/280 and OD260/230 to check the quantity and purity. The sequencing libraries for transcriptome sequencing were made with 1.5 μg of total RNA by using the NEBNext® Ultra™ RNA Library Prep Kit from Illumina® (NEB, USA) following the manufacturer's instructions. Poly-T oligo-attached magnetic beads were used to purify the mRNA, and the mRNA was fragmented with divalent cations and heat using the NEBNext First Strand Synthesis Reaction Buffer. Fragments of 200-250 bp were purified with the AMPure XP system (Beckman Coulter, USA), and then 3 μL of the USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min, then 5 min at 95°C, and then PCR was carried out with a high-fidelity DNA polymerase along with the Universal PCR primers and Index (X) Primer. The Agilent Bioanalyzer 2100 system was used to assess the quality of the purified PCR products and library. The sequencing was carried out on an Illumina NovaSeq 6000 platform by Beijing Allwegene Technology (China), and paired-end 150 bp reads were made. About ~40 M raw reads for each sample were made.