title: GSM3063092: Stem RNA-seq; Salvia dorisiana; RNA-Seq
library_construction_protocol: Plant tissues were grinded and RNA was isolated with the RNeasy Plant Mini kit (Qiagen) according to the manufacturer protocol. Trichomes were grinded with a pestle and mortar while still frozen in N2, and transferred to a precooled Eppendorf-tube in liquid N2. Buffer RLT was added to the frozen trichomes and RNA was isolated further with the kit. RNA was treated with DNAse1 (Invitrogen). Of each isolation, one microgram total RNA was used for mRNA isolation and subsequent RNAseq library preparation following TruSeq Stranded mRNA Sample Preparation Protocol (Illumina). In short, mRNA was isolated using oligo dT beads and chemically fragmentized prior to first strand cDNA synthesis using random primers. Strand specificity was achieved by replacing dTTP with dUTP during second strand synthesis and the addition of Actinomycin D to the first strand master mix. Obtained double stranded cDNA fragments were used for 3'Adenylation and adapter ligation using different barcoded adapters. Adapter ligated cDNA was finally amplified using 15 PCR cycles. Quality control of final libraries was done using an Agilent Tapestation High Sensitivity D1000 Screen Tape assay. Final library quantification was performed using Qubit (Invitrogen). Equimolar amounts of libraries were pooled and diluted towards 6pM concentrations for paired end clustering on one lane of a flow cell V4 (Illumina). Final sequencing was performed on a HiSeq2500 instrument using a 126,7,126 flow cycle pattern. De-multiplexing of obtained sequences was done using CASAVA 1.8.1. software.
attributes.tag: GEO Accession
attributes.value: GSM3063092