title: GSM8484278: PK10-46; Homo sapiens; RNA-Seq
library_construction_protocol: Archived PDAC FFPE tumor samples were categorized as TLS-positive or TLS-negative based on H&E staining. Under a microscope, the tumor areas were marked using a marker on the bottom side of the H&E-stained slide glasses. The non-stained FFPE slides, which were serial sections of the H&E-stained sections, were used for tumor tissue collection. When scraping the non-stained FFPE tissues, the H&E-stained slide (with the marked tumor areas) was stacked beneath the non-stained FFPE slide. Using the markers visible through the stacked slides, only the tumor tissues were carefully scraped and collected from the non-stained FFPE sections. Total RNA was extracted from FFPE slides using Qiagen RNeasy FFPE kit following anufacturer's instructions (Qiagen, Hilden, Germany). RNA samples were quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) and RNA integrity was checked with 4200 TapeStation (Agilent Technologies, Palo Alto, CA, USA). rRNA depletion sequencing library was prepared by using QIAGEN FastSelect rRNA HMR Kit (Qiagen, Hilden, Germany). RNA sequencing library preparation uses NEBNext Ultra II RNA Library Preparation Kit for Illumina by following the manufacturer's recommendations (NEB, Ipswich, MA, USA). Briefly, enriched RNAs are fragmented for 15 minutes at 94 °C. First strand and second strand cDNA are subsequently synthesized. cDNA fragments are end repaired and adenylated at 3'ends, and universal adapters are ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified using Qubit 2.0 Fluorometer (ThermoFisher Scientific, Waltham, MA, USA) as well as by quantitative PCR (KAPA Biosystems, Wilmington, MA, USA).