Illumina Genome Analyzer IIx paired end sequencing
Illumina 76bp paired-end RNAseq of lynx pardinus liver. Almoradoux
The libraries were prepared using the mRNA-Seq sample preparation kit (Illumina Inc., Cat. # RS-100-0801) according to manufacturer's protocol with minor modifications. Briefly, 3 µg of total RNA was used for poly-A based mRNA enrichment selection using oligo-dT magnetic beads followed by fragmentation by divalent cations at elevated temperature. First strand cDNA synthesis by random hexamers and reverse transcriptase was followed by the second strand cDNA synthesis performed using RNAseH and DNA Pol I. Double stranded cDNA was end repaired, 3' adenylated and the 3'-T nucleotide at the Illumina adaptor was used for the adaptor ligation. The ligation products were size selected and purified using the E-gel agarose electrophoresis system (Invitrogen) targeting the library size in the range of ~300bp that will result in an insert size of about 200bp. Following the gel purification, the adapter ligated cDNA was amplified with 15 cycles of PCR.