GSM7384753: human_RLT_region_14; Homo sapiens; RNA-Seq
The slides were incubated with blocking Buffer W (200 ?L/slide, NanoString) for 30 min in the humidity chamber at room temperature after hybridization.Each slide was stained with corresponding morophology markers. After immunofluorescent staining, the slides were visualized using the GeoMx® DSP instrument to select regions of interest (ROIs). To acquire representative regions, ROI selection was performed by an expert pathologist. The pathologist was blinded to the group assignment during the ROIs selection. Based on fluorescent staining, each ROI was segmented into corresponding areas of interest (AOIs) - CD68+ regions, CD3+ regions, and CD20+ regions. Subsequently, each AOI was exposed to UV light and photocleaved oligos were aspirated from the solution into the wells of a collection plate. Collected photocleaved oligos were PCR amplified with the corresponding GeoMx® Seq Code Primer Plate and Master Mix (NanoString). PCR products were pooled and cleaned with AMPure XP beads (Agilent, Santa Clara, California, USA) twice to obtain the libraries. The quality and concentration of libraries were assessed using a high sensitivity DNA Kit (Agilent) and Bioanalyzer. Subsequently, libraries were sequenced on an illumina sequencing platform (Hiseq or NovaSeq) with standard workflow specifications (dual-indexing and paired-end reads (2?×?27?bp)).