sequencing of neurons isolated from mouse and human postmor-
tem tissues down to single cells. We utilized LCM as it enables
precise isolation of individual cells from postmortem tissues. As
tissue sections used for the procedure remain intact it preserves
the positional information of each captured cell. LCM can success-
fully be applied to tissues from any embryonic or postnatal stage. It
is particularly advantageous for adult animal tissues and human
postmortem samples that do not easily allow for tissue dissociation.
Smart-seq2 is a widely used method for single-cell RNA sequencing
with increased sensitivity and reproducibility compared to other
methods [2]. Furthermore, Smart-seq2 allows for a single tube
reaction with direct lysis of live cells and subsequent cDNA library
preparation, which we have adapted to cells isolated by LCM. Our
newly developed method LCM-seq enables spatial transcriptomic
profiling down to the single cell level, thus enabling the deduction
of gene expression in intact tissues at a significantly improved
resolution.2 Materials
Generalnote.Clean all work benches, instruments and equipment
with RNaseZAP and wipe with wet tissue (H 2 O or 70% ethanol).
Use certified RNase/DNase-free materials and molecular biology
grade reagents whenever available. Use filter tips. Wear gloves at all
times when handling materials and change gloves regularly. Clean-
ing gloves regularly with RNaseZap wipes is recommended, partic-
ularly before touching LCM slides. Keep samples as cold as possible
at all times and minimize handling to avoid degradation and con-
tamination. Keep dedicated reagents, materials, and equipment for
RNA work separately whenever possible.2.1 Tissue
Dissection
- 5% avertin (2,2,2-tribromoethanol) in PBS.
- 7 mL PE Pasteur pipette, cut to a spoon.
- 2-methylbutane (Isopentane), approximately 10 mL.
- Metal container for freezing reagent.
- Styrofoam box with dry ice.
- Dissection tools.
- RNaseZap wipes.
- Nuclease-free 1.5 mL tubes and aluminum foil for tissue
storage, labeled and precooled on dry ice.
9. 80 C freezer.
2.2 Tissue
Cryosectioning
- Cryostat, 20 C.
- RNaseZap wipes.
96 Susanne Nichterwitz et al.