F1 BurXSf_3w_r3
GROWTH: F1 crosses (Col-0 x Bur-0, Bur-0 x Col-0,  Col-0 x Sf-2, Sf-2 x Col-0, and Bur-0 x Sf-2) were grown under long photoperiod conditions (16 hrs. light and 8 hrs. dark) at 16ĄC, and leaves from either 2-week or 3-week old plants were harvested for total RNA-sequencing. RNA extraction: RNA was isolated with TRIzol¨ Reagent (#15596-018, Ambion¨) according to manufacturerŐs instructions. Contaminating genomic DNA was removed with the Turbo DNA-freeTM kit (#AM1907, Ambion¨), then TURBO DNase was inactivated with DNase Inactivation Reagent and centrifuged at 10,000 x g for 2 min. 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).
SUB2558358
X832