TruSeq targeted RNA sequencing of homo sapiens: CTCL skin
TruSeq targeted RNA sequencing of 284 genes of interest for Cutaneous T-Cell Lymphomas (CTCL) expression analysis. Total RNA extraction using Rneasy FFPE kit, quantified on a Nanodrop and RNA integrity numbers obtained on BioAnalyzer. Custom oligonucleotides probes for TruSeq (284 genes) were designed. Reverse transcription was then performed on total RNA. Targeted probes were then hybridized to cDNA and washed with Steptavidin beads and temperature was decreased to allow annealing. cDNAs with annealed probes were then subjected to nucleeotide chain extension and ligation using Illumina Extension and Ligation Mix 4. 29 cycles of PCR were then performed using TruSeq DNA polymerase. Produced sequencing libraries were pooled in 6 groups for sequencing on the Illumina MiSeq platform.