Jan 20, 2023
CUMC-TMC-10X-Gene-Expression
- skg 1
- 1Columbia University
- Cellular Senescence Network (SenNet) Method Development Community
Protocol Citation: skg 2023. CUMC-TMC-10X-Gene-Expression. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl4j398lo5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 20, 2023
Last Modified: January 20, 2023
Protocol Integer ID: 75641
Keywords: optimal gene expression testing condition, gene expression, cdna sample, tissue optimization, cumc, qpcr with kapa library quantification kit, cortex, gene, kapa library quantification kit, agilent bioanalyzer, qpcr
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.
Troubleshooting
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.