Thymus vulgaris RNA-seq (Tv13)
the Illumina, Inc. (San Diego, CA) TruSeq RNA sample Preparation v2 kit was used according to the manufacturer's protocol with the following modifications. In brief, poly-A containing mRNA molecules were purified from 2 ug of total RNA using poly-T oligo attached magnetic beads. The purified mRNA was fragmented by addition of the fragmentation buffer and was heated at 94°C in a thermocycler for 4 min. The fragmentation time of 4 min was used to yield library fragments of 300-400 bp. First strand cDNA was synthesized using random primers to eliminate the general bias towards the 3' end of the transcript. Second strand cDNA synthesis, end repair, A-tailing, and adapter ligation was done in accordance with the protocols supplied by the manufacturer. Purified cDNA templates were enriched by 15 cycles of PCR for 10 s at 98°C, 30 s at 65°C, and 30 s at 72°C using PE1.0 and PE2.0 primers and with Phusion® High-Fidelity DNA polymerase (New England Biolabs, Ipswich, MA). Each indexed cDNA library was verified and quantified using a DNA 100 Chip on a Bioanalyzer 2100 then equally mixed by nine (from different genotypes). The final library was then quantified by real time PCR with the Kapa Biosystems (Wilmington, MA) Library Quantification Kit for Illumina Sequencing Platforms adjusted to 10 nM in water and sent to the GeT core facility (Toulouse, FRANCE, http://get.genotoul.fr/) for sequencing.