20 pooled glass eels from St. Lawrence estuary (RB sample)
Total RNAs were individually extracted from entire glass eels preserved in RNA later (Ambion) using PureLink™ RNA mini kit (Invitrogen). Thereafter, mRNA were selected using MicroPoly(A)Purist™ Kit and used as template for cDNA synthesis following SMART™ cDNA Technology (Clontech) protocol for library construction with a modified oligo-dT primer (5’-aagcagtggtatcaacgcagagttttcttttttcttttttn-3’). Amplification of cDNA samples was performed using Advantage 2 Polymerase Mix (Clontech) and SMART™ primer under the following parameters: 15 min at 37° followed by 5 min at 94° and (40 s at 94°, 1 min at 65° and 6 min at 72°) x 18 cycles. At least 2 µg of amplified cDNA were then purified for each individual using QIAquick PCR Purification Kit (Qiagen), before being fragmented by sonication. Fragments from 300 to 800 bp were selected and purified using the QIAquick Gel extraction Kit (Qiagen). The fragmented cDNA was then polished to obtain blunt-end cDNA molecules and phosphorylated at their 5’-termini by treating simultaneously 100ng of sample with 3U of T4 DNA polymerase and 3U of T4 polynucleotide kinase in T4 DNA Ligase buffer with 10mM dNTP and 100µg.ml-1 of BSA. All reagents were purchased from New England Biolabs. The mix was incubated 15 min at 12°, immediately followed by 15 min at 25° and 20 min at 75°. In order to remove enzymes, cDNA fragments were then extracted by phenol/chloroform and ethanol precipitation. Thereafter, samples were ligated with two unphosphorylated partially double-stranded adaptors (10µM) : the standard 454 B primer and the standard 454 A primer with a 10 bp long barcode extension at its 3’ end (see technical bulletin of Roche No. 005-2009). Each of the 20 individuals per sampling site was identified by a specific barcode held by the A primer. Adaptors were ligated to fragmented cDNA using 400U of T4 DNA ligase (New England biolabs) during 15 min at 25° followed by 20 min at 65°. After ligation, constructs were gel purified and amplified by PCR using the standard 454 primer A and 5'-biotin-labeled primer B under the following amplification parameters:  94° for 5 min followed by (94° for 40 s, 65° 1 min, 72° for 3 min) x 17 cycles and ended by 4 min at 72°. The final products were again column purified (Qiagen) and quantified using the Quant-iT Picogreen dsDNA Assay Kit (Invitrogen). For each sampling site, the 20 individually tagged libraries were pooled in equal amounts to obtain a mix containing 5 µg of cDNA. Fragments ranging from 350 to 900 bp were selected and purified to remove small fragments. The original protocol (MARGULIES et al. 2005) was then followed from the step of fragment immobilization onto streptavidin beads and each mix was sequenced on a half-plate of Roche GS-FLX DNA Sequencer.
Université Laval
Anguilla_rostrata_RB