Cryosections of OCT–embedded tissue blocks were transferred to PEN membrane glass slides and stained with cresyl violet acetate. Adjacent sections were H&E stained for pathology review. Laser capture microdissection was performed on a PALM MicroBeam microscope (Zeiss), collecting at least 1000 cells per compartment. RNA was extracted and libraries prepared using the Ovation RNA-Seq System V2 kit (NuGEN). Libraries were sequenced to a depth of 30 million, 100bp, single-end reads.
Protocol detail
Transcriptomic Profiling of Laser-Captured Cells
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Cells
Cresyl violet acetate
Cryosections
Laser capture microdissection
Membrane
Microscope
Palm
Rna seq
Tissue
Corresponding Organization : Columbia University Irving Medical Center
Other organizations : NYU Langone Health, University of California, San Diego, Psychogenics (United States)
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Protocol cited in 9 other protocols
Variable analysis
independent variables
- Tissue fixation and embedding method (OCT embedding)
- Staining method (cresyl violet acetate staining)
- Laser capture microdissection technique
- RNA extraction and library preparation
- Sequencing depth and read length
- Adjacent sections stained with H&E for pathology review
- Positive control: Not specified
- Negative control: Not specified
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