title: Population-level transcriptome sequencing of nonmodel organism Erynnis propertius and Papilio zelicaon
design: Library construction was performed by Express Genomics, Inc. (Frederick, Maryland, USA).  Poly(A)+RNA from the E. propertius total RNAs was isolated by two rounds of   oligo(dT) selection with oligo(dT) coated magnetic particles (Seradyn, Inc.).     From the poly(A)+RNA mRNA, cDNA libraries were constructed by using an oligo dT primer-adapter   containing a Not I site and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) to prime   and synthesize first strand cDNA. After the second strand was synthesized, the double stranded cDNA   was size fractionated (>1.4 kb) and cloned directionally into the Not I and Eco RV sites of the pExpress   1 vector. From one bulk ligation (300 ng of pExpress 1 vector, Not I-Eco RV cut, and 120 ng of Not I digested   cDNA per 120 microlitres of ligation), followed by electroporation into T1 phage resistant E. coli, primary   clones were produced.    Normalized cDNA libraries were produced from the primary cDNA libraries. Biotinylated   driver RNA produced from the T7 RNA polymerase promoter and single-stranded (ss) target DNA   produced from the F1 ori were hybridized to each other at a low Cot (concentration of driver   times the time of hybridization) value. The RNA:DNA hybrids were removed by phenol extraction   and the remaining ss target DNA was converted to double-stranded DNA (dsDNA) with a repair oligo   and Taq DNA polymerase. After  electroporation of the dsDNA into T1 phage resistant E. coli,   primary clones were produced.