TITLE: Irradiation and single-cell dissociation of hESCs and cortical spheroids
AUTHORS: Annika Martin, Hanqin Li, Dirk Hockemeyer

[DESCRIPTION]
This protocol describes the procedure of irradiation and single-cell dissociation of hESCs and cortical spheroids for MULTI-Seq barcoding and sequencing. 

Protocol Overview
A. Irradiation of hESC 
B. hESCs derived cortical spheroids

Note
A list of reagents and relevant vendor information can be found in the table listed under the materials tab.

[STEPS]
SECTION: Irradiation of hESCs
1. Culture hESCs with feeder-free system (dx.doi.org/10.17504/protocols.io.b4mcqu2w)
SECTION: Irradiation of hESCs
2. 3 days before irradiation, passage cells and let them grow to approximately 50% confluence.
SECTION: Irradiation of hESCs
3. On the day of irradiation, change media to hESC media + 10uM Rock Inhibitor (RI).
SECTION: Irradiation of hESCs
4. Using a discrete cesium source, irradiate cells in plates at desired dosage. We used 0, 0.5, 2, 5 and 10 Gy.
SECTION: Irradiation of hESCs
5. 24 hours post irradiation, dissociate cells by aspirating media and adding 1 mL of accutase per well of a 6-well plate. Return to incubator for 5 min to 10 min.
SECTION: Irradiation of hESCs
6. Resuspend cells using 10 mL of PBS- to dilute accutase. Spin down for 5 min at 300 x g. 

SECTION: Irradiation of hESCs
7. Aspirate supernatant and resuspend cells in 1 mL PBS- per well.

SECTION: Irradiation of hESCs
8. Label cells with MULTIseq oligos as described in MULTI-Seq Barcoding and Library Preparation protocol (dx.doi.org/10.17504/protocols.io.kxygx3xzkg8j/v1)  and proceed with sequencing.
SECTION: hESCs Derived Cortical Spheroids
9. 
 

Differentiate hESCs to Cortical spheroids as described in Cortical Spheroid Differentiation protocol (https://doi.org/10.17504/protocols.io.5jyl8po57g2w/v1.).
SECTION: hESCs Derived Cortical Spheroids
10. At 25 days in differentiation, two organoids per condition were transferred with 1.5 mL of media into a 1.5mL Cryovial with screw top. Using a discrete cesium source, irradiate organoids in vials at desired dosage. We used 0, 0.5, 2, 5 and 10 Gy. 
SECTION: hESCs Derived Cortical Spheroids
11. Post-irradiation, transfer both organoids per condition into one well of a 6-well low adherence plate with 2.5 mLadditional media. Add Rock Inhibitor (RI) to final concentration of  10uM.
SECTION: hESCs Derived Cortical Spheroids
12. 24 hours post irradiation, prepare 5 mL of Activated Papain Solution per irradiation condition:
SECTION: hESCs Derived Cortical Spheroids
12.1. To each 5mL of Dissociation Media, add 12 µL of 0.5M EDTA, 23ul of 0.1uM β-mercaptoethanol, 5 µL of 5.5mM L-Cystine solution, and 172 µL of papain suspension.
SECTION: hESCs Derived Cortical Spheroids
12.2. Transfer the solution to the 37 °C water bath for ~20 min to 30 minutes to activate. The solution will gradually go from cloudy to transparent.
SECTION: hESCs Derived Cortical Spheroids
12.3. Sterilize by passing the solution through a 0.22 μm syringe filter.
SECTION: hESCs Derived Cortical Spheroids
13. Collect cortical spheroids and sediment. Aspirate the media and rinse once with PBS+.
SECTION: hESCs Derived Cortical Spheroids
14. Add 5 mL Activated Papain Solution to each well and return to the incubator for ~ 45 min.
SECTION: hESCs Derived Cortical Spheroids
15. While dissociating, prepare 15mL Trypsin Inhibitor solution per 5 mL of Activated Papain Solution.
SECTION: hESCs Derived Cortical Spheroids
15.1. For each 15mL Trypsin Inhibitor Solution: Add 15 mg Trypsin Inhibitor to 15 mLof usual organoid media. Mix thoroughly and sterilize by filtering through a 0.22 μm syringe filter.
SECTION: hESCs Derived Cortical Spheroids
16. After 45 min of dissociation, add 5 mL of Trypsin Inhibitor solution to each well and very gently triturate with a 5mL strippette to dissociate. Avoid excessive pipetting and bubbles.
SECTION: hESCs Derived Cortical Spheroids
17. Strain mixture through a 70μm cell strainer into a 50mL conical tube.
SECTION: hESCs Derived Cortical Spheroids
18. Use remaining 10 mL Trypsin Inhibitor Solution to rinse the well to collect any cells left behind, then pass this through the same 70μm strainer to release any cells still stuck in the mesh.
SECTION: hESCs Derived Cortical Spheroids
19. Spin down cells at 300 x g for 5 min and aspirate the media.

SECTION: hESCs Derived Cortical Spheroids
20. Resuspend dissociated cells in 4 mL 0.2% BSA in PBS+ with RI per condition, strain through a 40um mesh cell strainer and FACS sort for desired number of live cells and to remove debris.
SECTION: hESCs Derived Cortical Spheroids
21. Spin down live cells at 300 x g for 5 min. Resuspend in 0.2% BSA in PBS+ with 10uM RI in appropriate volume according to single-cell sequencing protocol, count cells, and proceed to single-cell sequencing according to manufacturer protocol.