title: GSM3370072: Col-0 Heat AM Rep 4; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: Aerial tissue was harvested and frozen in liquid nitrogen prior to RNA extraction. Purified total RNA was extracted from 4 replicates of mock treated and heat shocked plants using Qiagen RNeasy Plant Minikits, following manufacturer protocols. Samples were diluted in order to acquire two micrograms of RNA in preparation for mRNA isolation. mRNA was separated using the NEBNext Poly(A) Magnetic mRNA isolation kit (New England Biolabs). Before library preparation, mRNA samples were heated to 95°C for 15 minutes to achieve 150-200 base pair fragment sizes. NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) was then used to generate libraries for sequencing. cDNA was synthesized with random hexamers.  After performing end repair and adaptor ligation steps, Agencourt AMPure XP beads (Beckman Coulter) were used following recommended ratios in order to isolate 150-200 bp fragments and remove adaptors. After, end repair and adaptor ligation size selection was performed with AMPure beads to isolate 150-200bp fragments and removed adaptors. PCR library enrichment for fifteen cycles was performed. The libraries were run on Agilent Bioanalyzer high sensitivity DNA chip after a 1:4 (or 1:10 if the concentration is too high) dilution. NEBNext Library Quant Kit for Illumina was used to measure library quantity and quality. Finally, the libraries were diluted to 10 nmol/ul concentrations before sequencing.
attributes.tag: GEO Accession
attributes.value: GSM3370072