title: Illumina HiSeq 2000 sequencing; RNA-Seq of arabidopsis with mutant against WT control
design: RNA-Seq of arabidopsis with mutant against WT control
library_construction_protocol: Frozen leaves from 14-day-old plants were collected in liquid nitrogen The plants were grown in a growth room at 22°C under a 12-h light/12-h dark cycle, with a light intensity of 120 mol m–2 s–1 provided by cool-white fluorescent bulbs. Frozen leaves from 14-day-old plants were ground in liquid nitrogen and their RNA was extracted using Trizol reagent (Thermo Fisher Scientific). The total RNA samples are first treated with DNase I to degrade any possible DNA contamination. Then the mRNA is enriched by using the oligo(dT) magnetic beads (for eukaryotes) or by removing rRNAs from the total RNA (for prokaryotes). Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments (about 200 bp). Then the first strand of cDNA is synthesized by using random hexamer-primer. Buffer, dNTPs, RNase H and DNA polymerase I are added to synthesize the second strand. The double strand cDNA is purified with magnetic beads. End reparation and 3'-end single nucleotide A (adenine) addition is then performed. Finally, sequencing adaptors are ligated to the fragments. The fragments are enriched by PCR amplification.
identifiers.namespace: Photosynthesis Research Center, Key Laboratory of Photobiology, Institute of Botany, Chinese Academy of Sciences
identifiers.id: E-MTAB-7904:Mutant_s