title: GSM8457227: GCSCs 002; Homo sapiens; RNA-Seq
library_construction_protocol: Based on the inclusion-exclusion criteria, tumor tissues from three gastric cancer patients were collected and prepared into single-cell suspensions. Cells were labeled with antibodies against two gastric cancer stem cell surface markers, CD54 and CD44. The labeled cells were sorted by flow cytometric sorting technique to obtain CD54+ CD44+ double-positive cell subpopulation and CD54- CD44- double-negative cell subpopulation of gastric cancer stem cells (GCSCs) and non-gastric cancer stem cells (Non-GCSCs), respectively. Total RNA was extracted from GCSCs and Non-GCSCs with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Total RNA quality was assessed using agarose gel electrophoresis and quantified using a NanoDrop2000 spectrophotometer (Thermo Fisher Scientific, USA). The Agilent Bioanalyzer 2100 (Agilent Technologies, USA) was used to verify RNA integrity. The fragmented RNA was used as a template for first strand cDNA synthesis, using random primers (Random Hexamers).The second strand of cDNA was synthesized using DNA polymerase and the double stranded cDNA was purified with magnetic beads (AMPure XP; Beckman Coulter,USA).Then addition of adenlylate 3′ ends, connection sequencing adapters, selection library size, PCR amplification and checking library quality were performed in turn. The whole transcriptome sequencing project requires the construction of two sequencing libraries simultaneously, including a strand-specific library for rRNA removal and a small RNA library. The constructed library was sequenced using Illumina high-throughput sequencing platform according to standard protocol (Genesky Biotechnologies Inc., Shanghai, China). This study obtained the expression landscape of mRNAs, miRNAs, circRNAs, and lncRNAs from the same samples in the discovery set and conducted subsequent bioinformatics analysis.The cDNA library was sequenced on an Illumina NovaSeq 6000 sequencing platform to generate read pairs of 150 bp length.