3.6.1 Reverse
Transcription and PCR
Amplification
- Preheat thermocycler to 72C.
- Prepare master mix 1 and 2 and place the metal tube rack on
ice. - Retrieve LCM-isolated cell samples from 80 C.
- Spin down the samples in a microcentrifuge and transfer into
PCR tube strips (optional,seeNote 16). - Add 2.1μL of master mix 1 (do not pipette up and down to
avoid loss of material) and spin down. - Incubate for 3 min at 72C to open the secondary structures of
the RNA molecules. - Spin down and place back on ice (oligo dTs anneal during snap
cooling). - Add 5.45μL of master mix 2 and spin down.
- Initiate the RT reaction by heating samples at 42C for 90 min.
Subsequently incubate samples at 50C for 2 min and at 42C
for 2 min, and repeat this for 10 cycles. Finally, incubate
samples at 70C for 15 min. The RT reaction takes approxi-
mately 2.5 h after which samples are spun down. - Prepare the PCR master mix 3, add 15μL per sample and spin
down. - Use the following program for PCR; 3 min at 98C, then
18 cycles of (20 s at 98C, 15 s at 67C, and 6 min at 72C)
(seeNote 17). End the PCR with a 5 min incubation at 72C.
The PCR is approximately 2.5 h in length. Spin down the
samples.
3.6.2 Bead Purification 1. Take the 19.5% PEG bead solution out of the fridge at least
20 min before starting the purification.
- Transfer cDNA (25μL) into a 96-well plate.
- Mix the 19.5% PEG bead solution well by vortexing.
- Add 25μL of the 19.5% PEG bead mix to the cDNA, set the
pipet to 50μL and mix well by pipetting up and down ten
times, while being careful not to produce bubbles. - Cover the plate and incubate at room temperature for 8 min.
- Place the plate (covered) on a magnetic stand and leave for
5 min. - Take off the supernatant while being careful not to disturb the
beads. - Wash by adding 200μL of 80% ethanol toward the opposite
side of the beads, shake gently, then remove ethanol, repeat
once.
Spatial Transcriptomic Profiling Using LCM-seq 105