3.6.3 Tagmentation and
Sequencing Index Ligation
- Preheat the PCR machine to 55C.
Prepare the tagmentation master mix 4 on ice, mix well (see
Notes 19and 20 ). - Transfer 1 ng of cDNA per sample to a PCR tube and adjust the
volume with ddH 2 O to 10.6μL. - Add 9.4μL of master mix 4 (to a total volume of 20μL), pipet
up and down five times. - Incubate at 55C for 5 min.
- Immediately add 5μL 0.2% SDS (seeNote 21) (pipet up and
down five times) and incubate for 5 min at room temperature
to strip enzymes from the DNA. - Prepare PCR master mix 5 on ice.
- Place tubes back on ice.
- Add 5μL index primers (5μL each, diluted 1:5 in ddH 2 O).
Choose a unique combination of two sequencing indexes
indices (N7xx and S5xx) for each sample if they are to be
sequenced on the same lane. Be careful not to confuse lids of
different tubes to avoid cross contamination. - Add 15μL of PCR master mix (master mix 5), pipet up and
down (total volume 50μL now). - Use the following program for the final enrichment PCR;
3 min at 72C, 30 s at 95C, then 10 cycles (seeNote 22)
of 10 s at 95C, 30 s at 55C, and 30 s at 72C. Finalize with
5 min incubation at 72C and set the PCR machine to hold at
4 C thereafter. - Spin down samples and transfer to a 96-well plate for bead
purification.
3.6.4 Bead Purification 1. Take the 24% PEG bead solution out of the fridge at least
20 min prior to initiating the purification.
- For protocolseeSubheading3.6.2, use 24% PEG beads now
and add 50μL of bead mix (1:1) instep 3.
3.6.5 Pooling of Samples
for Sequencing
- Measure concentration of sequencing libraries on Qubit.
- Pool equal amounts of DNA for sequencing in one lane (see
Note 23).
4 Notes
- The duration of the freezing process depends on the size of the
tissue and can be judged by the color change. It takes approxi-
mately 7 s for an adult mouse brain to freeze and 3 s for a spinal
cord. Do not submerge tissues too long as they may crack.
Spatial Transcriptomic Profiling Using LCM-seq 107