F2 Sample 26 (Technical Replicate 3)
GROWTH: 162 samples (a subset of the samples released under GEO series number GSE92568) consisting of single F2 plants of a cross between Arabidopsis thaliana accessions Algutsrum (8230) and TDr-9 (6195) were generated. F2 seeds were stratified for 5 days in 0.1 % agarose at 4ĄC. Plants were germinated and grown 2 plants per pot in 8 cm2 pots on Sunshine LA4P Mix (Sun Gro Horticulture, Bellevue, WA, USA) at 16ĄC constant temperature with a 16 hour light photoperiod. Plants were harvested at the 9 true leaf stage between 7.5 and 8.5 hours into the photoperiod. RNA extraction: Individual frozen plants were ground in liquid nitrogen using a mortar and pestle and RNA was isolated using PureLink Plant RNA Reagent (#12322-012, Invitrogen, Carlsbad, CA, USA) following the manufacturers instructions. Precipitated RNA was resuspended in RNAsecure Resuspension Solution (#AM7010, Ambion; Life Technologies, Carlsbad, CA, USA) following manufacturers guidelines to prevent RNA degradation. Contaminating genomic DNA was removed by incubating samples with Turbo DNase (#AM2238, Ambion) for 15 min at 37ĄC. Finally, RNA was precipitated using 2.5 vol 100% ethanol, 0.1 volume 5M Ammonium Acetate and 1ľl glycogen (5 mg/ml) and resuspended in RNase-free water. LIBRARY CONSTRUCTION: We prepared libraries using the SENSE Total RNA-Seq Library Prep Kit (Lexogen) according to the manufacturerŐs instructions with the following specifications: 200 ng of total RNA were used as input; the sample was incubated at 37 ĄC for 110 min. in the reverse transcription and ligation step; after the second strand synthesis step, 27 ľl of Purification Solution were added in the first purification step to increase the fraction of inserts < 200 nt; and 16 cycles were used during the amplification step. Libraries were validated with a Fragment AnalyzerŞ Automated CE System (Advanced Analytical), concentrations were determined for all fragments between 200 and 700 bp, and pooled in two sets at equimolar concentration for 96-multiplex sequencing. Libraries were sequenced on an Illumina HiSeqŞ 2500 Analyzer using manufacturerŐs standard cluster generation and sequencing protocols in 100 bp SE mode. DATA PROCESSING: Removal of adapter contamination was performed with BBDuk from the BBMap package (v35.10; B. Bushnell, http://sourceforge.net/projects/bbmap/). The first 11 and the last 6 nucleotides of every read were further removed with Seqtk (H. Li, https://github.com/lh3/seqtk). Finally, reads shorter than 50 bp were removed with Trimmomatic (v0.33; Bolger et al., 2014).
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