tube holder for inspection to ensure that no cells remain in
the cap.- Keep time from thawing of slides for LCM to freezing the
samples below 2 h. - For small numbers of collected cells, perform RT and PCR
amplification in the collection tube to avoid loss of material.
For a larger amount of cells collected, it is ok to transfer
samples to PCR strips to facilitate handling. - The number of PCR cycles depends on the input material and
should be determined for each project. 18 PCR cycles are
sufficient for 1–150 LCM dissected motor neurons
(> 200 μm^2 area). - The time to air-dry the bead pellet is variable (5–15 min). Do
not over-dry the magnetic beads. If some pellets are dry before
others, already add the elution buffer to these while leaving the
plate on the magnetic stand. - Pipette PEG slowly as it is very viscous and a final concentra-
tion in the 8–10% range is critical. - Preparation of sequencing libraries can be achieved using the
Nextera XT DNA Library Preparation Kit (Illumina) using the
manufacturer’s recommendations. However, to reduce cost we
have instead used a recombinant Tn5 enzyme produced in
house as described in detail in (3). The volume of Tn5 needed
for tagmentation can vary between batches of the enzyme and
we have observed a loss of enzymatic activity over time. Test
and adjust the volume for every new batch or when tagmenta-
tion is becoming inefficient. - Be precise when preparing and pipetting the 0.2% SDS solu-
tion. A slightly higher concentration of SDS may inhibit the
PCR reaction. - The number of cycles can be increased to 12 if input DNA is
much less than 1 ng. - The amount of samples that will be sequenced in one lane
depends on the sequencing platform and the sequencing
depth desired (i.e., a specific biological question). Only pool
samples that have unique combinations of sequencing indices
(N7xx and S5xx). For recommended combinations of indices
see Illumina’s guideline for sample pooling.
References
- Nichterwitz S, Chen G et al (2016) Laser capture
microscopy coupled with Smart-seq2 for precise
spatial transcriptomic profiling. Nat Commun
7:12139. doi:10.1038/ncomms12139 - Picelli S et al (2013) Smart-seq2 for sensitive
full-length transcriptome profiling in single
cells. Nat Methods 10:1096–1098. doi:10.
1038/nmeth.2639- Picelli S et al (2014) Tn5 transposase and tag-
mentation procedures for massively scaled
sequencing projects. Genome Res
24:2033–2040. doi:10.1101/gr.177881.114
110 Susanne Nichterwitz et al.