Sep 06, 2023
KAPP-Sen TMC: 10x Visium CytAssist FFPE
- William F. Flynn1,
- Paul Gabriel Jr.1,
- Emily C. Soja1,
- Grace K. Frohock1,
- Jessica D. S. Grassmann1,
- Allison D. McNeilly1,
- Sandra L. Daigle2,
- Greggory Perry2,
- Anahita Amiri1,
- Shannon Bessonett1,
- Diane Luo1,
- Marie Jacques Martine Seignon1,
- Elise T. Courtois1,
- Paul Robson1,3,4
- 1The Jackson Laboratory for Genomic Medicine, Farmington, CT, USA;
- 2The Jackson Laboratory, Bar Harbor, ME, USA;
- 3Department of Genetics and Genome Sciences, University of Connecticut School of Medicine, Farmington, CT, USA;
- 4Institute for Systems Genomics, University of Connecticut, Farmington, CT, USA
- Cellular Senescence Network (SenNet) Method Development Community
- KAPP-Sen TMC
Protocol Citation: William F. Flynn, Paul Gabriel Jr., Emily C. Soja, Grace K. Frohock, Jessica D. S. Grassmann, Allison D. McNeilly, Sandra L. Daigle, Greggory Perry, Anahita Amiri, Shannon Bessonett, Diane Luo, Marie Jacques Martine Seignon, Elise T. Courtois, Paul Robson 2023. KAPP-Sen TMC: 10x Visium CytAssist FFPE. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm3ew6l3p/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: September 02, 2023
Last Modified: September 06, 2023
Protocol Integer ID: 87260
Keywords: SenNet KAPP-Sen TMC
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Abstract
The Visium CytAssist Spatial Gene Expression workflow assays RNA levels in intact formalin-fixed paraffin embedded (FFPE) tissue sections. It uses probes targeting the whole transcriptome. Each Visium CytAssist Spatial Gene Expression Slide contains Capture Areas with barcoded spots that include oligonucleotides required to capture gene expression probes.
Tissue sections are processed with Visium Spatial Gene Expression assay performed by the Single Cell Biology Core at The Jackson Laboratory for Genomic Medicine.
Deparaffinization, H&E, Decrosslinking
Deparaffinization, H&E, Decrosslinking
Visium CytAssist Spatial Gene Expression for FFPE – Deparaffinization, H&E Staining, Imaging & Decrosslinking: CG000520 Rev B
CG000520_Demonstrated_Protocol_VisiumCytAssist_Deparaffin_H_E_RevB.pdf3.1MB Library Preparation
Library Preparation
Visium CytAssist Spatial Gene Expression Reagent Kits User Guide: CG000495 Rev E
CG000495_VisiumCytAssist_GeneExpressionUserGuide_RevE.pdf31MB Library concentration was quantified using a Tapestation High Sensitivity DNA ScreenTape (Agilent) and fluorometry (Thermofisher Qubit) and verified via KAPA qPCR.
Library Sequencing
Library Sequencing
Libraries were pooled for sequencing on an Illumina NovaSeq 6000 200 cycle flow cell using a 28-10-10-90 read configuration, targeting 100,000 read pairs per spot covered by tissue.
1000000019358_17_novaseq-6000-system-guide.pdf1.5MB FASTQ Generation
FASTQ Generation
Illumina base call files for all libraries were converted to FASTQs using bcl2fastq v2.20.0.422 (Illumina).