Single-cell RNA sequencing

mod. Sato et al. 2009 (https://doi.org/10.1038/nature07935) and Haber et al. 2017 (https://doi.org/10.1038/nature24489 )

Reagents:

• 20mM EDTA in PBS (Ca/Mg-free)
• TrypLE express, no phenol red (Thermo Fisher 12604013)
• 5% FBS (VWR10802-772) in PBS (Ca/Mg-free)
• FACS buffer: 5% FBS in DMEM/F12, HEPES, no phenol red (Thermo Fisher 11039021) à filter
• DNase (Worthington Biochemical LK003172)

Before you start: 

• prepare 20 mM EDTA in PBS (Ca/Mg-free)
• prepare 5 % FBS in PBS
• prepare FACS buffer
• prepare DNase
• warm up TrypLE express

Procedure:

1. Dissect the small intestine in cold PBS                
2. Rinse and flush with cold PBS                             
3. Cut open longitudinally and slice ~5 mm
4. Incubate the tissue in 5 mL of cold 20 mM EDTA-PBS (Ca/Mg-free PBS) in a 15 mL tube (I used LoBind Protein tubes (Eppendorf 0030122216), but I didn’t test if regular tubes would result in a lower yield) with gentle rocking in cold room            
5. After 20’, shake the tissue, settle the fragments down, and collect supernatant: fraction 1
6. Incubate the remaining tissue in new  5 mL of cold 20 mM EDTA-PBS (Ca/Mg-free PBS) with gentle rocking in cold room
7. After 20’, shake the tissue, settle the fragments down, and collect supernatant: fraction 2
8. Centrifuge the supernatant (fraction 1+2 combined) 300 g x 3’
9. Optional: repeat steps 6-7 for more fractions if desired (see Figure 2—figure supplement 1 A)
10. Resuspend and wash in 5 mL cold 5 % FBS in PBS (Ca/Mg-free)
11. Centrifuge 300 g x 3’
12. Resuspend and wash in 5 mL cold 5 % FBS in PBS (Ca/Mg-free)
13. Centrifuge 300 g x 3’
14. Dissociate in 5 mL of TrypLE express + 250uL of DNase (~112U/mL) in 37c water bath x1-2’
15. Centrifuge the supernatant 300 g x 3’
16. Resuspend and wash in 5 mL cold 5% FBS in PBS (Ca/Mg-free)     
17. Centrifuge 300 g x 3’
18. Resuspend the pellet in 2 mL FACS buffer with DNase
19. Add TOPRO3 and Calcein Violet 1/10,000
20. Pass through 70-um filter and 40-um filter, wash the filter with additional 1 mL FACS buffer
21. FACS (collect tdTomato+, Calcein Violet+, TO-PRO-3- cells) à scRNA-seq