Next Generation Sequencing Facilitates Quantitative Analysis of shControl and shLINC00205 Gastric Cancer Cell Lines Transcriptomes

Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare the transcriptome differences between knockdown LINC00205 and negative control gastric cancer cells. Methods: Total RNA from the BGC823 cells with LINC00205 knockdown and negative control were isolated using RNeasy mini kit (Qiagen, Germany). Paired-end libraries were synthesized by using the TruSeq RNA Sample Preparation Kit (Illumina, USA) following TruSeq RNA Sample Preparation Guide. Briefly, The poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. 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 HiSeq X-ten (Illumina, USA). The library construction and sequencing was performed at Shanghai Biotechnology Corporation. STAR (version:2.7.6a) was used to map the cleaned reads to the human GRCh38 reference genome with two mismatches 1. Then ,we ran Subread/featureCounts (version:2.0.2) with a reference annotation to generate gene expression counts and FPKM values for known gene model. Results: We identified a set of differentially expressed genes after LINC00205 knockdown. The result of GO and KEGG enrichment analysis showed high confidence of genes enriched in the function of cell proliferation, cell cycle, and cell adhesion, consistent with the canonical molecules of these pathways significantly modulated. Conclusion:The results suggested that LINC00205 could affect the key regulated pathway that controls the progression and metastasis of GC.