Design: cDNA production (first strand): First-strand cDNA poly(A) synthesis was performed using the SuperScript III First-Strand kit (18080-051, Invitrogen). One microgram of total RNA was reverse-transcribed using the oligo(dT)20 primer provided by the kit in a reaction volume of 20 µL. A control reaction was also performed without the Superscript reverse transcriptase enzyme to monitor genomic contaminations. cDNA production (second strand):Full-length cDNA synthesis: First-strand cDNA synthesis was performed with the PrimeScript Reverse Transcriptase (Clontech Laboratries) using the SMART (Switching Mechanism at 5' end of RNA Transcript) PCR technology (Clontech Laboratories), but following a modified protocol suggested by Roche Diagnostics to optimize sequencing using the 454 FLX Sequencing technology. Total RNA was reverse-transcribed using a modified CDS III oligo(dT) (Invitrogen Life Technologies): 5'-TAGAGACCGAGGCGGCCGACATGTTTTGTTTTTTTTTCTTTTTTTTTTN-3' (N corresponds to the mix of A, G, or C). At the 5'-terminus of the template, a poly(C) tail is added to the cDNA using the terminal transferase activity of the reverse transcriptase. The SMART V oligonucleotide provided with the kit hybridizes to the poly(C) tail to form an RNA/DNA hybrid: 5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3'. Full-length LD PCR (long distance polymerase chain reaction):For the LD PCR reaction, we used the Advantage 2 PCR Kit (Clontech Laboratries). Only sscDNAs that have the 5'-SMART anchor can be used as a template for the LD PCR reaction, thus ruling out eventual genomic DNA contamination. For each time course (30 min, 3 h, 6 h, and 9 h), we performed 5 × 100 µL PCR reactions. To each 100-µL mix, we added 2 µL of first-strand cDNA reaction, 10 µL of Advantage Buffer10×, 2 µL of 10 mM dNTP mix, 2 µL of 10 µM 5'-SMART PCR primer, 2 µL of 10 µM Modified CDSIII 3'-PCR Primer, 2 µL of Advantage Polymerase mix 50×. The reaction volume was completed with 80 µL of RNase/DNase-free H2O. PCR were performed as follows: 1 min at 95°C (20 sec at 95°C, 6 min at 68°C) for 23 cycles. The optimal number of cycles was determined previously as 23 cycles by comparing different numbers of cycles, that is, 20, 23, 26, and 29 cycles. To assess the quality of the dscDNA sample, we loaded 5 µL of each sample onto a 1.1% agarose gel, resulting in a smear from 100 bp to 3.5 kb. The dscDNA was cleaned and concentrated using the PureLink PCR Purification Kit (Invitrogen Life Technologies). Samples were eluted with 40 µL of H2O. cDNA purity was measured using the 260/280 nM ratio against 10 mM Tris (pH 7.5). LD PCR primers:5'-SMART V PCR: 5'-AAGCAGTGGTATCAACGCAGAGT-3'; Modified CDSIII 3'PCR (N corresponds to the mixture of A, C or G): 5'-TAGAGGCCGAGGCGGCCGACATGTTTTGTCTTTTGTTCTGTTTCTTTTN-3'.
Submitted by: CNRS (CNRS-IGS)
Study:
Mimivirus Transcriptional Landscapeshow Abstracthide AbstractMassively parallel pyrosequencing of polyadenylated RNA fractions of A. castellanii cells infected by mimivirus at various time during the infection.
Sample:
Generic sample from Acanthamoeba polyphaga mimivirusLibrary:
Name: MIMI_T12
Instrument: 454 GS FLX
Strategy: FL-cDNA
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Spot descriptor:
5 forward
Runs:
1 run, 76,310 spots, 21.5M bases, 42.3Mb