BMC Genomics 2007, 8:363 http://www.biomedcentral.com/1471-2164/8/363 e.g. [1], including the abundant but low complexity tRNAs and rRNAs and the increasingly studied popula- tions of various snRNAs, scRNAs, snoRNAs, telomeric RNAs, vRNAs, and microRNAs [2-7]. They lack represen- tation of the mRNAs of organelles – mitochondria and chloroplasts – for which polyadenylation may be a signal for degradation [8,9]. They lack representation of mRNAs that are not poly-adenylated or lose their polyA tails, but are nevertheless translated [10-13]. The recent call for a more systematic examination of the entire transcriptome – RNomics [14] led to much greater interest in ncRNAs and a variety of wet and computational approaches to their identification [reviewed in [15,16]]. Our purpose here was to develop a library construction method that would result in a more complete representa- tion, in useable form, of the transcriptome. We reasoned that self-subtraction [17,18], which equalizes the repre- sentation of different sequences through reassociation kinetics, should work as well as poly A+ selection for reducing the frequency of the abundant but low-complex- ity rRNAs and tRNAs, without eliminating them, or any other polyA- RNAs, from cDNA libraries. We describe here the method and show that informative, random-primed, directional, and more completely representative cDNA libraries can be made from partially degraded total RNA. Results and discussion RNA preparation Since a method for the production of high-quality, more fully representative cDNA libraries from even difficult samples was sought, three very different RNA sources were chosen: 48 hour zebrafish (Danio rerio) embryos, field- collected 36 hour embryonic amphioxus (Branchiostoma floridae), and isolated 3rd instar fruitfly (Drosophila mela- nogaster) larval brain and eye discs. Total RNA was extracted from these samples with TRIzol (Invitrogen, manufacturer's protocol). Contaminating genomic DNA was completely removed from aliquots of the RNAs by digestion with 1 U RNase-free DNase (NewEngland Biolabs)/ug RNA in the manufacturer's buffer for 30 min- utes at room temperature. 1 µg of RNase free glycogen (Roche) was then added and the sample re-extracted with Trizol (Methods-1). Total RNA was partially hydrolyzed in 100 mM (Na)CO3, pH 10.0, for 20 min. at 60°C. This resulted in a population of 100–1300 nt RNA fragments (Methods-2). cDNA synthesis Double-stranded (ds) cDNAs were synthesized from the partially-degraded total RNA by a standard Gubler-Hoff- man replacement procedure. Primer and template were annealed by mixing 5 µg of the partially hydrolyzed, DNA-free RNA with 0.7 µg of the 5'-phosphorylated, directional, 1st-strand primer (DRP1) (Fig. 1A), heating to RFaignudroem1directional cDNA synthesis Random directional cDNA synthesis. A. The first strand cDNA primer (DRP1) contains: a 5' phosphorylated buffer sequence devoid of A bases (1), an AscI site (2), a 5 nt ran- dom sequence (3), and a 3' T residue (4). By design, priming should begin only at an A in the RNA template. B. The non- phosphorylated lone-linker LL1 consists of the the two com- plimentary oligonucleotides LL1F and LL1R. It has one blunt end and one non-adhesive staggered end. LL1 can therefore ligate only to one strand of the cDNAs and in only one ori- entation. The remaining nick in the second strand is removed by preincubating the cDNAs before the first PCR reaction at 72°C for one minute to strip off the non-ligated strand of the linker and regenerate the sequence by extension from the 3' end of the cDNA (lower grey). 70°C for 10 minutes, and quenching briefly on ice. The first strand was synthesized by incubating the annealed mix with 200 units of Superscript II reverse transcriptase (Invitrogen) for one hour at 45°C in the manufacturer's buffer containing 10 mM DTT and 0.5 mM dNTPs. The second strand was synthesized by adding 90 µl H2O, 32 µl 5× 2nd strand buffer [100 mM HEPES pH 6.9 (buffered with KOH), 50 mM KCl, 25 mM MgCl2, 50 mM (NH4)2SO4], 6 µl dNTPs (5 mM each), 1 µl ®NAD (10 mM), 2 µl E. coli DNA ligase (6 U/µg), 6 µl 0.1 M DTT, 4 µl E. coli DNA polymerase I (10 U/ul), and 1 µl E. coli RNase H (2 U/µl). The reaction mixture was incubated for 16 hours at 16°C. Double-stranded cDNAs were then blunt-ended by adding 10 units of T4 DNA polymerase (New England Biolabs) and continuing the incubation for 10 minutes. The blunt-ended, double-stranded cDNAs (yields were between 1.2 and 1.6 µg) were then extracted with phenol:chloroform (1:1), precipitated with ammo- Page 2 of 7 (page number not for citation purposes)