Insights into shell deposition in the Antarctic bivalve Laternula elliptica: gene discovery in the mantle transcriptome using 454 pyroseqeuencing
Mantle RNA was extracted from 24 animals using a modified TRI reagent protocol. After homogenization in Tri Reagent (Sigma) and chloroform extraction, the samples were subjected to a lithium chloride precipitation step. RNA was precipitated using a 1:1 isopropanol:saline solution (1:1 0.8 M sodium citrate and 1.2 M NaCl) and after resuspension, the RNA was subjected to a further precipitation using 250 mkl 7.5 M LiCl. The extractions were further cleaned using RNeasy mini kit columns (Qiagen, Crawley, Sussex, UK) following manufacturer instructions in order to eliminate LiCl and salt residues. 5mkg of RNA was PCR amplified using the protocol described in [78] prior to preparation for the 454 run. Samples were nebulised at 30psi for one minute and subsequently purified with Ampure (Agencourt) to produce fragments 300 bp and above. The ends were polished and the 454 titananium adapters containing specific MID sequences were attached. Fragments containing both a and b adapters were selected and quantified . Libraries were amplified by emulsion PCR, beads recovered and enriched and placed on a picotiter plate for sequencing by the 454 procedure. 12 Samples were MID tagged for each run (of 2).