Single-cell RNA sequencing

1. Transfer  handpicked islets (approximately 5,000 IEQs) into 15mL conical tube. 

2. Add 10mL of 1xPBS w/o Ca2+, Mg2+ (Rockland, MB-008). Centrifuge for 2 min at RT, 180 xg. Aspirate the supernatant.

3. Add 1mL of warm (37°C ) 0.05% Trypsin (Invitrogen, 25300054) to the islets. Pipette up and down with p1000. 

4. Incubate at 37°C for 9 min, or until cells are in single cells. Pipette up and down at t=7 min, 4 min, 2 min, 0 min. 

5. Stop the trypsin reaction by adding 1mL of 100% FBS (Hyclone, SH3091003) to the dissociated islets and pass cells through BD FACs tube with strainer top (Corning 352235)

6. Use 1mL of 100% FBS to rinse the tube and pass through the strainer.

7. Transfer cells to 15mL conical. Centrifuge 4 min, 400 xg. 

8. Remove the supernatant and wash cells with PBS with 10% FBS. Centrifuge for 4 min, 400 xg. 

9. Wash the cells with PBS with 10% FBS and centrifuge for 4 min, 400 xg. Remove the supernatant.

10. Count cells using a countess chamber.

11. For the scRNAseq, do the final resuspension in 10% FBS in PBS and adjust the volume to make the final suspension 1000 cells/microliter. Filter the cells one more time prior to loading them onto the 10X Genomics chip. 

1. Older HPAP samples (specifically donors HPAP-001 to HPAP-019) were processed using C1 Single-Cell mRNA Seq HT IFC and Reagent Kit v2 (product ID: 101-4964) which has been discontinued.

2. All current samples (HPAP-019 onwards) were processed using the Chromium Single Cell 3’ Reagent Kit.

3. Unfortunately, since 10x Genomics is going to stop manufacturing the above kit, we will be using the new kit Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1. For this protocol we target a 5000 cell recovery.