Prepare brain slices

Follow your favourite slicing protocol. This is what I do.

1. Deeply anaesthetize the mouse thiopental.

2. OPTIONAL If the mouse is over 40 days old, perfuse transcardially with 5mL ice cold bubbled ACSF.

3. Extract the brain and place in the vibratome in ice cold bubbled cutting solution.

4. Cut 350 um slices of the region of interest.

5. For each slice, briefly rinse in bubbled room temperature ACSF, then incubate in bubbled ACSF at 34 C for 30 min.

6. Afterwards, keep slices in bubbled ACSF at RT.

Collecting cells

I typically carry on with cell collection for 6-8 hours after slicing.

7. Transfer a slice to the microscope and perfuse with bubbled ACSF at RT

8. Load a freshly pulled pipette with internal solution, apply positive pressure and inspect the tip under the microscope. Typically, I go for 3-4 um diameter, or 1-2 MOhm.

9. Approach the cell of interest with positive pressure, then apply negative pressure and monitor the aspiration. If possible, acquire video of the process for future reference.

10. Once the cell is harvested, withdraw the pipette and break the pipette tip against the bottom of a PCR tube containing Smart-seq2 lysis buffer.

CRITICALPull the pipette out of the lysis buffer quickly to avoid drawing the lysis buffer into it by capillary action.

11. Immediately freeze the tube on dry ice or at -80 C.

PAUSESamples can be stored at -80 C for several weeks.

Smart-seq2 RT and preamp

12. Prepare Smart-seq2 RT master mix.

13. Take samples from the -80 and incubate at 72 C for 3 min in a thermocycler, then place on ice immediately.

CRITICALPlacing samples on ice reduces secondary structure formation.

14. With the samples on ice, add 6.35µL of the Smart-seq2 RT master mix. Do not pipette up and down.

15. Briefly spin the samples down.

16. Run the RT reaction in a thermocycler: 42 C for 90 min, then 10 cycles of 50 C for 2 min and 42 C for 2 min, followed by 70 C for 15 min.

Allegedly cycling at 50/42 C can be skipped when in a rush without a substantial loss of yield. However, this may introduce a bias against transcripts with secondary structure.

PAUSEcDNA can remain in the thermocycler over night at 10 C.

17. Add 15µL of the PCR master mix to each sample.

18. Run PCR preamp: 98 C for 3 min, then 20 cycles of 98 C for 20 s, 67 C for 15 s, 72 C for 6 minc, followed by 72 C for 5 min.

Because some of the RNA is lost in the aspiration procedure (remaining stuck to the glass?), I increased the number of cycles compared with the published Smart-seq2 protocol. These parameters typically yield 1ng/µL of amplified cDNA for good samples.

19. Purify with 20µL of AMPure XP beads, eluting in 15µL of elution buffer (10mM Tris-HCl, pH 8.5). Water is fine too.

20. OPTIONAL Check the yield by running 1µL of the sample on a DNA HS chip.

PAUSEPurified cDNA can be stored at -20 C for all eternity.

21. Proceed to Nextera XT library prep. If not checking the yield, 1µL of the sample usually works.