Generic sample from Ciona intestinalis
Ciona intestinalis
Random-primed SL- PCR consists of reverse-transcription primed by random hexamer sequences linked 3' to an arbitrary 25-nt anchor sequence (AN), followed by PCR using the AN sequence as a leftward primer and a 23-mer primer terminating in the 16-nt SL sequence, which is present at the 5'-end of trans-spliced mRNAs, as the rightward primer (SL primer). Because one of the amplification primers includes the SL sequence, SL-PCR products are expected to be 5'-segments of trans-spliced mRNAs, and because the initial reverse transcription was random-hexamer-primed, all trans-spliced mRNAs are expected to be represented, regardless of length. We applied random-primed SL-PCR to RNA isolated from tailbud stage embryos of Ciona intestinalis.     Adult Ciona intestinalis collected under permit in San Diego, California USA were maintained in a closed marine aquarium under constant illumination to induce gamete accumulation. Tailbud embryos were prepared by mixing eggs and sperm dissected from several individual adults and incubating in sea water at 18 C for 12 hr. RNA was isolated from embryos collected by low speed centrifugation by homogenization in 50mM Tris-HCl buffer pH 7.5, 50 mM NaCl, 10 mM EDTA, 0.5% SDS, digestion with proteinase K, phenol/chloroform extraction, and ethanol precipitation.    RNA was reverse-transcribed (8 min at 30 C followed by 50 min at 42 C) using Invitrogen Superscript II reverse transcriptase and buffer components, and random-hexamer primers linked 3' to an arbitrary 25-mer anchor DNA sequence termed AN (5'-GAATTCTACCTCAGAGGAGTCATAT-3'). The resulting cDNA was amplified by SL-PCR using the 25-mer AN primer and a 23-mer SL primer (5'-GGATCCGATTCTATTTGAATAAG-3') whose last 16 bases correspond to the Ciona spliced leader (SL) sequence. Thermostable DNA polymerase and buffer components were from the Clontech Advantage 2 PCR Kit. Thermal cycling conditions were 94 C, 1 min; [94 C, 30 sec; 57 C, 30 sec; 68 C, 30sec] x 25 cycles ; 68 C 2 min. SL-PCR products were recovered from the reaction using the Qiagen QIAquick PCR Purification Kit, which is expected to recover DNA molecules $100 bp, and following ethanol precipitation separate aliquots were analyzed by ultra high-throughput 454 pyrosequencing using the 454/Roche GS-20 sequencer and by T/A cloning in the pGEM-T vector (Promega) to assess the product length distribution. T/A cloning product insert sizes were determined by PCR amplification with primers based on vector T7 and SP6 promoter sequences. Of 19 plasmids recovered, 4 had no (or undetectably short ,&lt;50 bp) inserts and the remainder had inserts ranging from ~100 - 850 bp (median 350 bp, mode 500 bp, mean and standard deviation 403 ± 232 bp).