CUMC-TMC-10X-Gene-Expression
Shaunice Grier
Abstract
Post Mortem Human Fresh Frozen Dorsolateral Prefrontal Cortex tissues are cryo - sectioned onto 10X Visium slides.
Tissue Optimization is performed and determines optimal Gene Expression testing conditions.
cDNA samples are processed and generated into libraries. Libraries are qc'd for size and concentration with the Agilent Bioanalyzer 2100, Qubit Fluorometer and qPCR with KAPA Library Quantification Kit. QC'd libraries are submitted for sequencing.
Steps
Tissue QC
Section
H&E Stain
Image
Assess
Tissue Optimization
Permeabilization & cDNA Synthesis
Tissue Removal
Gene Expression
cDNA Synthesis
Second Strand Synthesis & Denaturation
cDNA Amplification & QC
Visium Spatial Gene Expression Library Construction
Sequencing
Submitted libraries are evaluated for size distribution on the Fragment Analyzer and quantified using Picogreen and qPCR with the Universal KAPA Library Quantification Kit. Library pools are loaded between 1.2 – 1.5 nM using the standard NovaSeq workflow as outlined in Illumina’s NovaSeq 6000 system guide.
