Analysis of expressed sequence tags by pyrosequencing: Identification and characterization of delta, sigma, omega, epsilon, theta and zeta class glutathione S-transferase (GST) genes in the aquatic midge Chironomus riparius (Diptera: Chironomidae) fourth instar larvae
RNA extraction and cDNA synthesis for pyrosequencing  The larvae (4th instar) were collected and total RNA samples were isolated using TRIZOL Reagent (Invitrogen Life Technology). The RNA samples (A260/A280>1.8) were collected and mRNA was purified from the pooled total RNA (500 mkg) by binding to oligo (dT) cellulose twice (Poly (A) Purist, Ambion).  For first strand synthesis, 10 mkl of the purified mRNA (5 mkg), denatured at 65 degrees C for 10 min in a RNase free tube, rapidly chilled on ice, mixed with 5 mkl of 10x first-strand buffer, 5 mkl of 100 mM DTT, 5 mkl of dNTPs (2.5 mM each), 5 mkl of Oligo dT20 (50mkM), 2.5 mkl of Strata Script Reverse Transcriptase (200 U/mkl) in a 50 mkl reaction volume.  First strand cDNA was synthesized at 42 degrees C for 60 min and cDNA synthesis and heat inactivated at 70oC for 15 minutes and the tubes were placed on ice.  For second strand cDNA synthesis, H2O (61 mkl), 100 M Tris-HCl, 20 mkl of second-strand buffer, dNTPs (6 mkl, 10 mM each), DNA polymerase I (4 mkl, 10 U/mkl), and RNase H (2 mkl, 1.5 U/mkl) were mixed with the first strand synthesis reaction and incubated at 16C for 150 min. For end blunting, 23 mkl of blunting dNTP mix, 2 mkl of cloned pfu DNA polymerase (2.5 U/mkl) was incubated with the second strand synthesis reaction at 16 degrees C for 5 min. The cDNA was purified using QIAquick(R) PCR Purification Kit (QIAGEN) in a final elution volume of 50 mkl.    Library preparation, emulsion PCR, sequencing run and assembly of sequences  Approximately, 1mkg of the final PCR product DNA was used to generate DNA library for Genome Sequencer FLX Titanium (Roche, Mannheim, GE). The fragments ends were polished (blunted), and short two adapters were ligated onto both ends and DNA sequencing was performed following protocols for the Genome Sequencer GS FLX System (Roche Diagnostic).  The reads were assembled using the GS De Novo Assembler (http://www.454.com/products-solutions/analysis-tools/gs-de-novo-assembler.asp).