Instrument: Ion Torrent Proton
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: All FFPE tissue slides were stained with hematoxylin and reviewed by an experienced gynecological pathologist. The selected lesions from whole tissues were excised by laser capture microdissection. For LCM, the FFPE tissues were sectioned and placed on slides with polyethylene terephthalate membrane (Leica Microsystems Inc., IL, USA). LCM was performed using a Leica AS LMD laser microdissection system (Leica Microsystems Inc.) according to the manufacturer's instructions. An RNeasy FFPE kit (Qiagen, Valencia, CA, USA) was used to isolate total RNA from FFPE tissues according to the manufacturer's instructions. Sample emulsion PCR, emulsion breaking, and enrichment were performed using an Ion PITM Template OT2 200 Kit v3 (Life Technologies, Part #4488318 Rev. B.0) according to the manufacturer's instructions. Multiple barcoded libraries were combined with equal molar ratios for one Ion PITM v2 chip. Two-pooled Ion AmpliSeqTM Exome libraries were loaded onto a single Ion PITM v2 chip. Five-pooled Ion AmpliSeq™ Transcriptome libraries were loaded onto a single Ion PITM v2 chip. Subsequent emulsion PCR and enrichment of the sequencing beads of the pooled libraries were performed using the Ion OneTouchTM system (Life Technologies) within approximately 7 h, according to the manufacturer's protocol. Finally, 520 Flows sequencing was done on the Ion PITM v2 chip using Ion PITM Sequencing 200 Kit v3 (Life Technologies, Part #4488315 Rev. B.0) on the Ion ProtonTM sequencer (Life Technologies).