TITLE: CRISPR tagging of the EEA1 gene in H9 ES cells for Endo-IP
AUTHORS: Jiuchun Zhang, Harper JW

[DESCRIPTION]
This protocol describes a method for knockin of a 3X flag tag onto the N-terminus of the EEA1 gene in human H9 ES cells using CAS9 and an ultramer oligonucleotide 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. 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 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 sequencing by MiSeq
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 sequencing by MiSeq
18.1. 10-14 days post sorting, split cells in 2 sets; 1 set for sequencing (Miseq) 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 sequencing by MiSeq
18.2. Sequencing by Miseq (Illumina):
	-  Make lysate by incubating cells in PBND solution (50mM KCl, 10mM Tris-HCl, pH 8.3, 2.5mM MgCl2-6H2O, 0.45% NP-40, 0.45% Tween-20) with 0.2% protease K (Qiagen) for 5 min at 37°C, and transfer lysate to a PCR plate and run the program:
	    55°C for 30min
	    99°C for 15min
	    Hold at 4°C
-         Perform the first round of PCR to amplify the region of interest using gene-specific primers
	For each sample, we do a 10 μl PCR.
                2x Master Mix (NEB Next Ultra II Q5): 5 μl
                Primer 1 [5'-ACACTCTTTCCCTACACGACGCTCTTCCGATCTaccccgagtagtgagtggc](100 μM): 0.07 μl
                Primer 2 [5'-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTcacgtgctgccagaaagac] (100 μM): 0.07 μl
                Water: 3.86 μl
                Lysate: 1 μl
	Run a 20-cycle PCR using appropriate annealing temp and extension time using the following sequence. 
1.    98°C, 30 sec                 
2.    98°C, 10 sec
3.    64°C, 15 sec
4.    72°C, 30 sec
5.    Go to b for additional 19 cycles
6.    72°C, 2 min
7.    4°C, hold
Note: Primer 1 and Primer 2 amplify the genomic region of interest. Each primer contains partial Illumina adaptor serving as anchor sites for the 2nd round of PCR.
-          Perform the second round of PCR by universal primers that contains attachment site for the flow cells and index sequences
	For each sample, we do a 10 μl PCR.
  2x Master Mix (NEB Next Ultra II Q5): 5 μl
	Primer CDI 666 (100 uM): 0.1 μl
  Primer index (10 μM): 1 μl
   Water: 2.9 μl
  Product from 1st round of PCR: 1 μl
 Run a 20-cycle PCR using appropriate annealing temp and extension time. 
Subject to MiSeq using manufacturers protocols (MiSeq Kit #MS-102-3001).