title: GSM5172100: Arabidopsis roots - ctl1-2 R3; Arabidopsis thaliana; RNA-Seq
library_construction_protocol: Roots from 14 days-old seedlings grown on plates  were harvested and immediately frozen in liquid N2. Three independent replicates were used for the transcriptome assay. Root material was ground with mortar and pestle in liquid N2. 50 -100 mg of ground material was used for RNA extraction using the RNeasy plant mini Kit (Qiagen). Extraction was performed according to the user manual provided by the supplier (RNeasy Mini Kit handbook, Fourth edition, June 2012, Qiagen). Extraction buffer RLC with 10 µl/ml β-mercaptoethanol freshly added was used to extract the RNA. An on-column DNA digestion (RNase-Free DNase Set, Qiagen) was performed. Before elution of the RNA the column was incubated 1 min with 30 µl RNAse-free water to resolve the RNA. 3'mRNA-libraries were prepared using the 3'mRNA-Seq library Prep Kit (QuantSeq, Lexogen). The manual from the supplier was followed with the following modifications. At least 1 µg RNA was used as input. For first strand synthesis of cDNA the incubation time at step 4 was increased to 60 min. For library indexing and amplification 13-17 amplification cycles of the given PCR were used depending on the amount of input RNA. 17 µl of the purified library was transferred to a fresh tube. The finished libraries were stored at -20° C until quality control and pooling.