title: GSM5468543: RNA-seq_DF1_R1.fastq.gz; Homo sapiens; RNA-Seq
library_construction_protocol: For RNA extraction, 10μm sections were cut from each sample (the first 5 sections of each FFPE block were discarded to avoid contamination). The sections were stored in sterile 2ml eppendorfs (one eppendorf for each sample regardless the number of sections) and stored at -20˚C until RNA extraction. Total RNA was extracted via a spin-column based method, using the AllPrep DNA/RNA FFPE Kit (Qiagen, Valencia, CA, USA), according to the manufacturer's recommendations. Initial deparaffinization step was performed with 640μl of Zymo Research deparaffinization solution (Catalog # D3067-1-20) per sample. RNA was finally eluted in 15-25 μl RNasefree water and stored at -80˚C until library preparation. cDNA libraries were prepared with 300ng total RNA input  using KAPA Stranded RNA-Seq Kit with RiboErase (catalogue number #KK8483, KAPA Biosystems, Wilmington, MA), according to the manufacturer's specifications. As a high percentage of each sample was already in the 100-200 bp range, the fragmentation step was omitted. In brief, the workflow included rRNA depletion, cDNA synthesis, end repair to generate blunt ends, A-tailing, adapter ligation and PCR amplification. PCR amplification consisted of an initial denaturation step at 98°C for 45 sec, followed by 15 amplification cycles (denaturation at 98°C for 15 sec; annealing at 60°C for 30 sec; and extension at 72°C for 30 sec) and final extension at 72°C for 5 min.