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Sample GSM7758252

Query DataSets for GSM7758252

Status

Public on Jan 25, 2024

Title

human_RLT_region_176

Sample type

SRA

Source name

reactive lympoid tissues

Organism

Homo sapiens

Characteristics

tissue: reactive lympoid tissues
region cell_type: CD68

Extracted molecule

total RNA

Extraction protocol

The slides were incubated with blocking Buffer W (200 μL/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)).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 3000

Description

4tonsils + 15-1b | 018 | CD68

Data processing

The GeoMx® NGS Pipeline is used to process FASTQ sequencing files from Nanostring GeoMx libraries to produce digital count conversion (.dcc) files. Once complete, all DCC files are packed into a zip file and available to download. This zip file can be uploaded into the GeoMx Digital Spatial Profiler (DSP) system for quality control and normalizaton.
Raw reads were trimmed, stitched, aligned, and deduplicated to transform digital counts data with unique targets. AOIs with fewer than 10,000 raw reads or sequencing saturation <50% were filtered out of the analysis. AOIs with less than 5% of all target genes (18,000+) and target genes that do not achieve the limit of quantitation were removed. The data was then processed with the Q3 normalization method for all the remaining targets. Q3 normalization divides the counts in one segment by the 3rd quartile value for that segment, then subsequently multiplies that value by the geometric mean of the 3rd quartile values of all segments. Q3 normalization rescales the gene expression data such that all segments have similar gene expression ranges. It reduces variance from segment size, segment cellularity, and other technical factors. Underlying the Q3 method is an assumption that, despite biological variation, segments should have similar gene expression count distributions. Thus, Q3 is only appropriate when a probe panel is large and diverse, such as one that targets the full transcriptome.
Assembly: hg38
Supplementary files format and content: The file named processed data (DSP_gene_matrix) showed the genes expression of all DLBCL tissues and 12 tonsil samples, sequencing by illimina hiseq3000.
Supplementary files format and content: The file named processed data2 (DSP_gene_matrix) showed the genes expression of the rest of tonsil samples, sequencing by illimina novaseq 6000.

Submission date

Sep 05, 2023

Last update date

Jan 25, 2024

Contact name

Min Liu

E-mail(s)

liuminnus@gmail.com

Organization name

National University of Singapore

Street address

13-02, 14 Medical Drive, Singapore