GSM9889636: Well-watered, polymer control, biological replicate 2; Phaseolus vulgaris; RNA-Seq
Total RNA was extracted from 100 mg of ground tissue using 1 mL TRIzol reagent. Samples were vortexed, mixed with 200 µL chloroform, incubated at room temperature for 3 min, and centrifuged at 12,000 rpm and 4°C for 15 min. The aqueous phase was transferred to a new tube, mixed with 500 µL isopropanol, incubated on ice for 10 min, and centrifuged at 12,000 rpm and 4°C for 10 min. RNA pellets were washed with 75% ethanol, centrifuged at 12,000 rpm and 4°C for 5 min, air-dried, resuspended in 35 µL DEPC-treated water, and incubated at 55°C for 10 min. RNA was further purified using a modified Plant Total RNA Extraction Miniprep System protocol. The RNA solution was mixed with 450 µL RX buffer and 230 µL 98% ethanol and loaded onto a spin column. Columns were centrifuged at 10,000 rpm for 1 min, washed with 500 µL WF buffer at 14,000 rpm for 1 min, and washed twice with 700 µL WS buffer. After a final centrifugation at 14,000 rpm for 3 min, RNA was eluted with 50 µL RNase-free water and stored at −80°C. RNA concentration and purity were assessed using a NanoDrop spectrophotometer, and RNA integrity was evaluated using an Agilent Bioanalyzer. Only samples with an RNA integrity number greater than 6.8 were used for sequencing. cDNA libraries were prepared by Novogene (HK) Co., Ltd. and sequenced on an Illumina NovaSeq 6000 platform to generate 150-bp paired-end reads.