TITLE: CRISPR tagging of the TMEM192 gene with 3xHA in H9 ES AAVS-NGN2 cells for Lyso-IP
AUTHORS: Jiuchun Zhang, Harper JW, Sharan Sharan Swarup

[DESCRIPTION]
This protocol describes a method for knockin of a 3X HA tag onto the C-terminus of the TMEM192 gene in human H9 ES AAVS1-NGN2 cells using CAS9 and a homologous recombination template 

[BEFORE_START]
Use ThermoFisher Kit to directly electroporate ESCs with Cas9 protein and sgRNA.
Works better than plasmid transfection.

[STEPS]
SECTION: preparing Cas9 RNP complex for electroporation
1. Add 10 µL to a sterile 1.5 ml tube. Add 6 µg. Then add 1.2 µg and 1 microgram TMEM192-3xHA homology arm plasmid (Addgene#175777). Pipet up and down to mix. Let it sit at Room temperature for 10 min. This is enough for 2 transfections (== one 6 well).

SECTION: preparing Cas9 RNP complex for electroporation
2. While waiting for the Cas9 to bind to sgRNA, individualize H9 ES AAVS-NGN2 cells with Accutase. Neutralize Accutase with 5x volume E8 with Rock inhibitor.
SECTION: preparing Cas9 RNP complex for electroporation
3. Count cells. You will need 2x105 for each transfection.
SECTION: preparing Cas9 RNP complex for electroporation
4. Spin down cells. Let it sit for a while so all the residue media can go down to the bottom of the tube. If the residue media is too much, take it out with a P200 pipet.
SECTION: preparing Cas9 RNP complex for electroporation
5. Resuspend cells to a concentration of 2x105 per 5 μl (ie 4x107 per ml) using buffer R. 
 

SECTION: preparing Cas9 RNP complex for electroporation
6. Prepare a 24 well matrigel coated plate. Add 0.5 mL-1 mL to the wells you will use. Add HAS (1:2500) to each well. Each transfection goes into one well.
SECTION: preparing Cas9 RNP complex for electroporation
7. Wipe the Neon pipet station with EtOH and place it inside the hood.
SECTION: preparing Cas9 RNP complex for electroporation
8. Add 3 mL to the neon tube. Place the tube inside the station. You should feel a click before the tube is securely seated in the station.
SECTION: preparing Cas9 RNP complex for electroporation
9. Use program 13 from the optimization tab for electroporation parameter. Program 9 should also work.
SECTION: preparing Cas9 RNP complex for electroporation
10. When everything is ready, mix 10 µL-11 µL with the Cas9+RNA containing R buffer. The final volume should be in the range of 21 µL-22 µL.
SECTION: preparing Cas9 RNP complex for electroporation
11. Take up a neon tip, pipet 10 µL and electroporate with program 13. 
 


SECTION: preparing Cas9 RNP complex for electroporation
12. If you see air bubble in the tip, take it out, push everything out of the tip and repipet the mixture.
SECTION: preparing Cas9 RNP complex for electroporation
13. If you see sparking during the electroporation, your efficiency will reduce significantly.
SECTION: preparing Cas9 RNP complex for electroporation
14. Once electroporation is complete, push everything into one well of a 24 well plate. Do not pipet up and down with Neon tip.
SECTION: preparing Cas9 RNP complex for electroporation
15. Repeat the same procedure with the same tip and the left over cell mixture. This is just a replicate.
SECTION: preparing Cas9 RNP complex for electroporation
16. Disperse cells evenly in the well and place cells in a low O2 incubator.
SECTION: preparing Cas9 RNP complex for electroporation
17. Put electroporated cells into low oxygen incubator for 2 days to help maintain viability. 
2. 
SECTION: Expansion of clones for analysis by immunoblotting
18. Sort cells into single wells of 96 well plates and keep cells in E8 medium + 10% Clone R2 (STEMCELL Technologies), and put cells into a low-oxygen incubator for 3-4 days till colonies are visible under the microscope, then move cells to a regular incubator. Change media with regular E8 every other day.
SECTION: Expansion of clones for analysis by immunoblotting
18.1. 10-14 days post sorting, split cells in 2 sets; 1 set for immunoblotting and the other for expansion. Keep cells in 10µM Rock inhibitor and 12.5µg/ml human serum albumin(HSA) while splitting. Consolidate cells while splitting if necessary.
SECTION: Expansion of clones for analysis by immunoblotting
18.2. SDS extracts from each well are subjected to SDS-PAGE (18% gel) and analyzed by immunoblotting with anti-TMEM192 antibodies (such as # PA5-118314 from ThermoFisher). For further validation, samples can also be probed with anti-HA antibodies (Biolegend; #901513). HA tagging causes a shift in the apparent molecular weight of TMEM192.