GSM2570307: S15C-1; Arabidopsis thaliana; RNA-Seq
Approximately 100 mg of tissue was used to extract total RNA from pulverized, frozen roots and shoots with an RNeasy Mini Kit (Qiagen) with on-column DNase treatment. mRNA was purified using Dynabeads Oligo (dT). Reverse transcription was performed using SuperScript III (Invitrogen, 18080-044) with second-strand synthesis with dUTPs, followed by cleanup, end repair, and dA-tailing. Universal Y-shaped adapter ligation was performed to enable sample multiplexing. Library amplification and multiplexing were performed using dUTP excision and amplification followed by PCR enrichment using 2x Phusion HF Master Mix (NEB, M0531L). Size-selection was performed using gel extraction and purification (Qiagen Gel Extraction Kit). Final quantity was determined using the DNA Bioanalyzer Chip and KAPA Library Quant qPCR.