title: Triticum turgidum Kronos transcriptome project, grain samples
design: Total RNA was extracted from frozen tissue using Spectrum™ Plant Total RNA Kit (Sigma), mRNA was purified using the Dynabeads mRNA Purification kit (Illumina) and transcribed to cDNA using SuperScript II Reverse transcriptase (Invitrogen) and DNA Polymerase I (Promega). The cDNA was purified using the PCR purification kit (Qiagen) and quality was assessed on the Bioanalyzer machine. The cDNAs were fragmented in a Covaris machine (10% duty cycle, Intensity: 4, Cycles per burst: 200, 80 sec), treated with end-repair enzymes: T4 DNA polymerase (Invitrogen), Klenow DNA Polymerase (Invitrogen) and T4 Polynucleotide kinase (Invitrogen), and adenylated with Klenow exo (3' to 5' exo minus) (Invitrogen).  Illumina PE adapters (Illumina Multiplexing kit, PE-400-1001) were ligated using the Quick Ligation kit (New England Biolabs) and purified with the minElute kit (Qiagen). DNA fragments were separated on a 2% agarose TAE gel; fragments with average size of 400 bp were extracted using Gel Excision tips (Gel Company) and purified using the Gel Extraction kit (Qiagen). The library was barcoded according to the instructions in the Illumina Multiplexing kit (PE-400-1001) with 12 cycles of PCR amplification. Final libraries were purified using Ampure beads in a 1:1 sample volume to bead volume ratio (Ampure). The quality of the libraries was assessed on the Bioanalyzer machine using High Sensitivity DNA kit reagents (Agilent).