Transcriptome Sequencing of GLYR1-positive human pancreatic ductal adenocarcinoma.

We performed transcriptome sequencing on GLYR1-positive and GLYR1-negative human pancreatic ductal adenocarcinoma (PDAC) to shed light on the transcriptional changes of GLYR1-positive PDAC. Overall design: The specimens were prepared by laser capture microdissection (LCM). In brief, surgically removed tissues were sectioned for H&E staining and LCM (Life technologies) was performed. Tumor tissues (stroma were excluded) were used for RNA extraction, and sequencing. Total RNA was isolated using RNeasy mini kit (Qiagen, Germany). Strand-specific libraries were prepared using the TruSeq® Stranded Total RNA Sample Preparation kit (Illumina, USA) following the manufacturer's instructions.Ribosomal RNA was removed from total RNA using Ribo-Zero rRNA removal beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under 94? for 8 min. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then go through an end repair process, the addition of a single 'A' base, and then ligation of the adapters. The products are then purified and enriched with PCR to create the final cDNA library. Purified libraries were quantified by Qubit® 2.0 Fluorometer (Life Technologies, USA) and validated by Agilent 2100 bioanalyzer (Agilent Technologies, USA) to confirm the insert size and calculate the mole concentration. Cluster was generated by cBot with the library diluted to 10 pM and then were sequenced on the Illumina NovaSeq 6000 (Illumina, USA).