- Place 0.2 mL PCR tubes into the collector and check tubes for
obvious contamination (change if contaminated) (seeFig. 3). - Place slides into the slide holder with the membrane facing
down. - Create “fast overview” of slides at 1.25magnification to
facilitate navigation. - Search for cells of interest at 5 or 10 magnification (see
Note 12and Figs.1 and 2). - Microdissect at 40 magnification: draw cutting outlines
closely to the cells to avoid contamination with other cell
types (seeNotes 3and 13 and Figs.1 and 2). - Capture cells into RNase/DNase-free 0.2 mL PCR tubes (dry
cap). - When sufficient numbers of cells have been collected, inspect
the collector and unload the tube holder (seeFig. 3). - Add a small volume of lysis buffer and pipette up and down a
few times to resuspend all cells (seeNote 14). - Spin down samples in a microcentrifuge for 15 s.
- Seal PCR tubes with Parafilm, label and snap-freeze on dry ice
(seeNote 15). - Store samples at 80 C until further processed.
3.6 cDNA and
Sequencing Library
Preparation
All of the following steps are carried out on ice if not otherwise
specified.Fig. 3PCR tube caps as seen in the tube holder of the LCM (Leica LMD7000).(a) Cap before and (b) after
collection of individual cells by laser microdissection at 5magnification.Note that the collected cells here
have relatively large soma sizes of> 200 μm^2 while smaller cells will be harder tosee.(c) Collected cells can
“hide” in uneven parts of the cap, especially when statics are high. If collection of a precise number of cells is
required, it is advisable to check the collector carefully at a higher magnification (20). Scale bar inb
(applicable toa, b)¼ 400 μm and inc¼ 50 μm
104 Susanne Nichterwitz et al.