Lupinus angustifolius cv P27255 Seed RNA-seq (singletons)
RNA isolated with 500 microL Trizol (incubated 15 minutes @ RT) from ~150 mg of matureing seed (20-26 days after anthesis) ground to a fine powder under liquid nitrogen.  After centrifugation at 12000g and 4 degrees Celcius for 10 min the supernatant was mixed with 200 microL chloroform and centrifuged again as above.  The upper phase was mixed with 300 microL 0.8M sodium citrate/1.2M NaCl and 300 microL isopropanol and incubated on ice for >10 min to precipitate total RNA.  RNA precipitate was centrifuged as above and washed twice in 75% ethanol, then dissoved in diethylpyrocabonated-treated water.  QC performed via BioAnalyzer and Qubit.  1 microgram of total RNA used to generated TruSeq RNA libraries and samples were pooled on a single lane of Illumina HiSeq1000 generating 100 bp paired-end reads with a ~250 bp insert size.  RNA-seq reads were trimmed for low quality <Q30, primer and adaptor sequences via Cutadapt 1.1 (overlap 10, times 3, minimum length 25).  Reads paired with discarded trimmed reads < 25 bp were retained as singleton reads.