GSM7062495: RNA_TLUSC4; Homo sapiens; RNA-Seq
FFPE sections were deparaffinized in mineral oil. Briefly, 800µL mineral oil (Fisher Scientific, #AC415080010) and 180µL Buffer PKD were mixed with the sections, Proteinase K was added for tissue digestion, and the sample was incubated at 56 °C for 15 min. Phase separation was encouraged with centrifugation, and the aqueous phase was chilled 3 min to precipitate RNA. After centrifugation for 15 min at 20,000 g, RNA-containing supernatant was removed for extraction, while DNA remained in the pellet. Nucleic acids were subsequently extracted using the AllPrep DNA/RNA Mini Kit (QIAGEN, #80204) according to the manufacturer's instructions. RNA was eluted in nuclease-free water and DNA in 0.5X Buffer ATE. Approximately 500 ng of FFPE RNA or 100 ng of fresh frozen RNA per sample was used for RNA library construction using the KAPA RNA Hyper library prep kit (Roche, Switzerland) per the manufacturer's instructions with minor modifications. Customized adapters with unique molecular indexes (UMI) (Integrated DNA Technologies, US) and Sample-specific dual-indexes primers (Integrated DNA Technologies, US) were added to each library. The quantity of libraries was measured with Qubit (Thermo Fisher Scientific, US) and quality measured by TapStation Genomic DNA Assay (Agilent Technologies, US). Equal amounts of each RNA library (around 500 ng) were pooled for hybridization capture with IDT Whole Exome Panel V1 (Integrated DNA Technologies, US) using a customized capture protocol modified from NimbleGen SeqCap Target Enrichment system (Roche, Switzerland). The captured DNA libraries were then sequenced on an Illumina HiSeq4000 with paired end reads (2 Å ~ 100 bp), at 50millions reads/sample.