GSM2553046: WT_1; Arabidopsis thaliana; RNA-Seq
seedlings treated with RNA later, RNA extracted with Ambion RNA kit (AM1919) The poly(A)+ RNA was reverse transcribed to cDNA (SuperScript Double-Stranded cDNA Synthesis Kit, Life Technologies). Random hexamers (New England BioLabs) were used for priming the first strand synthesis reaction and SPRI beads (Agencourt AMPure XP, Beckman Coulter) for purification of cDNA. Illumina compatible Nextera Technology (Illumina) was used for preparation of RNA-seq libraries, employing DNA fragmentation and tagging by in vitro cut-and-paste transposition. 60 ng of cDNA was used instead of DNA. After the tagmentation reaction, the fragmented cDNA was purified with SPRI beads. In order to add the Illumina specific bridge PCR compatible sites and enrich the library, limited-cycle PCR (5 cycles) was performed according to the instructions for the Nextera system with minor modifications. For bar coded libraries, 50 X Nextera Adaptor 2 was replaced with a bar coded Illumina-compatible Adaptors from the Nextera Bar Codes kit (Illumina) in PCR setup. SPRI beads were used for purification of the PCR-products and the library QC was evaluated by Agilent Bioanalyzer (Agilent).
GEO Accession
GSM2553046