GSM8768756: Localized PCa patient 70; Homo sapiens; RNA-Seq
RNA from 50ng of FFPERNAsamples.RNA from these samples were not fragmented previous to 1st strand cDNAsynthesis. The 1ststrand cDNA synthesis was performed using SMARTScribe reverse Transcriptase, for 90min. at 42°C, 10min. at 70°C and pause at 4°C. Afterwards Illumina Adapters and Indexes are added, performing a preamplification PCR (60sec at 94°, 5 cycles of 15sec at 98°C, 15sec a t55°C 30sec at 68°C; and pause at 4°C). Then Ribosomal cDNA is depleted with ZapR v2 and R-Probes v2. Finally, enrichment of libraries was achieved by PCR (60sec at 94°C; 13-19 cycles of 15sec at 98°C, 15sec at 55°C, 30sec at 68°C; and pause at 4°C). Sequencing libraries were prepared using “SMARTer Stranded TotalRNA-seq Kitv2–Pico Input Mammalian” kit (Takara Bio USA, Cat.# 634411), following “SMARTer Stranded Total RNA-seq Kitv2–Pico Input Mammalian User Manual (Rev.050619)”.