title: 454 sequencing of Sarracenia purpurea
design: After multiple extraction attempts using various kits and other wet lab techniques a successful  protocol was found using the Spectrum Plant Total RNA Kit (Sigma #STRN50-1KT). Only the  youngest unopened leaves were used as older tissues yield negligible RNA amounts. Certain   steps call for the use of RNase-free water; this was created by adding 0.1ml of  diethylpyrocarbonate to 100ml of water, incubating at 37°C for 12 hours and then autoclaving  for 15 minutes to remove trace amounts of the chemical. All instruments and surfaces were   cleaned thoroughly, treated with an RNase deactivating solution (100mM sodium hydroxide plus   .1% sodium lauryl sulfate) and wiped dry. The mortar and pestles were frozen with liquid   nitrogen for about 20 seconds prior to the start of tissue and maintained at a subzero temperature   throughout tissue grinding to prevent RNA degradation. A single total RNA prep of 100 mg of   young leaf tissue yielded on average 4-8 ug of high quality RNA. The extraction was repeated   40-50 times. We obtained approximately .25 mg of total RNA for each species.      The Oligotec mRNA Kit (Qiagen # 70022) was used to purify mRNA from 0.25 mg extracted   total RNA, according to the manufacturer’s recommendation. During the elution steps, 25ul of   OEB buffer was used in the initial step and 25ul in the follow up step for maximum mRNA  concentration in the smallest volume possible. mRNA quality was checked with a Bioanalyzer  (Agilent, Inc) and a NanoDrop 2000 (Thermo Scientific). For cDNA generation, the Evrogen   cDNA synthesis kit (SK001) was used with a modification to the kit’s 3’ primer. We used  approximately 26 ng of mRNA for each species.  Normalization of the cDNA was performed by using Evrogen Normalization Trimmer Kit  (NK001) in order to minimize the repetition of transcripts. This normalization protocol is based  on denaturing-reassociation of cDNA, followed by digestion with a duplex-specific nuclease  (DSN). The single-stranded cDNA fraction was amplified twice by sequential PCR reactions   according to the manufacturer’s protocol. A Qiagen MiniElut kit (#28006) was used to purify the  normalized cDNA and sterile water was used in the final elution in order to prevent the likely  interference of TE with 454 processing. Normalized cDNA in 100 ul sterile water was submitted  to the Georgia Genomic Facility (www.dna.uga.edu) for 454 sequencing in a Genome Sequencer  TM (GS) Titanium FLX instrument (Roche Diagnostics) employing a standard protocol. S.  psittacina and S. purpurea cDNAs were submitted at a concentration of 49 ng/ul and 45 ng/ul  respectively.