GSM3462539: Ctrl_late_6h_1; Branchiostoma lanceolatum; RNA-Seq
Embryos were frozen in liquid nitrogen after getting rid of the filtered sea water by centrifugation. Total RNA was extracted using the RNeasy Mini Plus Kit from QIAGEN following manufacturer's instructions. Disruption and homogenization were undertaken using TissueLyzer from QIAGEN. A library of template molecules suitable for high throughput DNA sequencing was created following the Illumina “Truseq RNA sample preparation low throughput” protocol with some modifications. Briefly, mRNA was purified from 2 µg total RNA using oligo-dT magnetic beads and fragmented using divalent cations at 94°C for 8 minutes. The cleaved mRNA fragments were reverse transcribed to cDNA using random primers, then the second strand of the cDNA was synthesized using Polymerase I and RNase H. The next steps of RNA-Seq library preparation were performed in an automated system using SPRIworks Fragment Library System I kit (ref A84801, Beckman Coulter, Inc) with the SPRI-TE instrument (Beckman Coulter, Inc). Briefly, in this system double stranded cDNA fragments were blunted, phosphorylated and ligated to indexed adapter dimers, and fragments in the range of ~200-400 bp were size selected. The automated steps were followed by PCR amplification (30 sec at 98°C; [10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C] x 15 cycles (6 hpt libraries) or 12 cycles (3 and 9 hpt libraries); 5 min at 72°C), then surplus PCR primers were removed by purification using AMPure XP beads (Agencourt Biosciences Corporation). DNA libraries were checked for quality and quantified using 2100 Bioanalyzer (Agilent).