Reads from pigmented (red) stem tissues (Source 6)
First and second strand cDNA synthesis was performed with 3 µg of the total RNA using Message Amp-II kit (Applied Biosystems/Ambion, Austin, TX, USA) following the manufacturers recommendations. A total of 10-12 ng of synthesized cDNA was amplified by in vitro transcription and the resulting 70-90 µg of antisense RNA (aRNA) was purified using Qiagen RNAeasy columns (Qiagen). A second round of cDNA synthesis was performed using the mRNA as template (20 µg). cDNA synthesis was performed as described above except that random primers (mostly hexamers) were used at the first strand synthesis stage. This procedure yielded approximately 10 µg of cDNA, subsequently purified using the DNA Clear Kit for cDNA purification (Ambion). cDNA was then treated with Ampure magnetic particles (Agencourt, Beckman Coulter, Danvers, MA, USA) to obtain fragments of 200 - 700 pb. The cDNA was prepared by using SMART PCR cDNA synthesis protocol (Clontech Laboratories, Inc., Mountain View, CA, USA) optimizing the conditions to obtain a high quantity of clean cDNA in a small volume. First strand synthesis was performed using 3 µg of total RNA for each sample with the use of Super Script II (Invitrogen) in a reaction mixture containing 50 mM Tris-HCl pH 8.3, 75 mM KCl, 6 mM MgCl2, 2 mM DTT. The retro-transcription reaction was primed with 3' SMART CDS Primer IIA (Clontech). The SMART II™A oligonucleotide (Clontech), which has an oligo(G) sequence at its 3' end, was used to create an extended template useful for the full-length cDNA enrichment via SMART™ technology. Second strand PCR synthesis was performed on a small aliquot (1/10th volume) of the primary template by using Advantage 2 Polymerase Mix (Clontech). The following thermal cycling program was applied: initial denaturation at 95°C for 60 s, followed by 15 cycles at: 95°C denaturation for 30 s; 55°C annealing for 30 s; 68°C extension for 6 min. All the PCR reactions were purified by using QIAquick PCR purification kit (Qiagen). Double stranded cDNA was quantified with a spectrophotometer (Nano Drop 1000, Thermo Scientific, Wilmington, DE, USA) and then concentrated by speed vacuum to a concentration of 500 ng/ul. The products were run on a 2% agarose gel to verify cDNA quality and fragment length. The main size distribution was within the 500 to 4,000 bp range. Approximately 5 µg of each cDNA sample were sheared via nebulization into small fragments, and then sequenced.