GSM4815449: ESSEX HL 9 (BBX32_1); Arabidopsis thaliana; RNA-Seq
Total RNA was isolated from four individual plants (4 leaves per plant) per treatment (8 in total)  using TRIzol reagent according to manufactuere's protocol  (Invitrogen), followed by DNAase1 treatment, further phenol extraction, ethanol precipitation and re-suspension in sterile distilled water after brief drying. Concnetration of RNA was deterimined initially using a nanodrop spectrophotomer. Quality of the RNA and its concentration was checked on a Agilent 2100 Bioanalyzer using RNA 6000 nanochip according to the manufacter's protocol. The 24 samples (represneting 6 goups of  biological replicates  were confirmed by quantitative real time PCR as having no significant differences between them in the cDNA levels of transcripts of a PP2A reference housekeeping gene. Samples were delivered frozen on dry ice within 24h to the sequence service provider (Novogene, Cambridge). This was conducted by Novogene (Cambridge, UK). The total RNA was enriched for mRNA using oligo-dT magnetic beads. After recovery of the enriched mRNA fraction, the RNA was fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamer primers and revrse transcriptase.After first-strand synthesis, a custom second-strand synthesis buffer  (Illumina) was added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation. The ds cDNA was purified from the  reaction solution, end repaired and a 3' A added. Adaptors were then ligated onto the tailed cDNA fragments. Sequencing adaptors were a standard Illumina design. Adaptor - fragments were size selected (250-300 bp insert size) and enriched by PCR amplification. Librar concentration was initially quantified using a Qubit 2.0 fluorimeter (Life Technologies). Insert size was checked on an Agilent 2100 Bioanalyzer and quantified using quantitative PCR.
GEO
GSM4815449