GSM6603068: Shoot_32SR_3; Arabidopsis thaliana; RNA-Seq
Total  RNA was isolated from Arabidopsis plants after temperature treatment  using the Trizol reagent and RNeasy columns (Qiagen). Quantity and quality measurements were taken using a NanoDrop spectrophotometer and bioanalizer. After the total RNA extraction and DNase I treatment, magnetic beads with Oligo (dT) are used to isolate mRNA. Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments. Then cDNA is synthesized  using the mRNA fragments as templates. Short fragments are purified and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. After that, the short  fragments are connected with adapters. After agarose gel electrophoresis, the suitable fragments are selected for the PCR amplification as templates. During the QC steps, Agilent  2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used in quantification and qualification of the sample library. At last, the library could be sequenced using  Illumina HiSeqTM 2000 or other sequencer when necessary