title: Illumina HiSeq 4000 paired end sequencing; RNAseq of combined LUAD and SCLC tumors to look at gene expression changes
design: RNAseq of combined LUAD and SCLC tumors to look at gene expression changes
library_construction_protocol: We identified 11 formalin-fixed paraffin-embedded (FFPE) tumors with combined LUAD and SCLC histology, from which independent isolation of both histological components was possible. We identified an additional 1 pre-transformation LUAD and 1 post-transformation SCLC cases for which tissue material was available. As controls we included a group of never-transformed LUADs and a set of de novo SCLC samples. RNA/DNA dual extraction from 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 minutes. Phase separation was encouraged with centrifugation, and the aqueous phase was chilled 3 minutes to precipitate RNA. After centrifugation for 15 minutes at 20,000g, 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.  RNA/DNA dual extraction from frozen tissue Frozen tissues were weighed and homogenized in RLT and nucleic acids were 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 EB.  Approximately 500ng of FFPE RNA or 100ng of fresh frozen RNA per sample were 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 500ng) 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).