TITLE: ATG9A-vesicle isolation protocol
AUTHORS: Daniel Bernklau, Justyna Sawa-Makarska, Julia Romanov

[DESCRIPTION]
This protocol details about the ATG9-vesicle isolation.

[BEFORE_START]
Hap1 cells were previously CRISPR engineered in our lab to contain ATG9A endogenously tagged on the
C-terminus with mEGFP-3C-FLAG.


[STEPS]
SECTION: Day1: Cell lysis and binding to Flag beads
2. Equilibrate Flag beads
Always pellet the beads with 4000 rcf, 4 °C cooled 5415R centrifuge for 3’.

SECTION: Day1: Cell lysis and binding to Flag beads
2.2. Wash 3x with MilliQ water
SECTION: Day1: Cell lysis and binding to Flag beads
2.3. Wash 3x with Vesicle Isolation Buffer.
SECTION: Day1: Cell lysis and binding to Flag beads
2.4. Prepare a 1 : 1 slurry in Vesicle isolation Buffer
SECTION: Day1: Cell lysis and binding to Flag beads
3. Cell lysis
Always work on ice
">> sample name" means: take a sample for Western Blot e.g. 10 µl sample + 2 µl 6xProtein loading dye
SECTION: Day1: Cell lysis and binding to Flag beads
3.2. Resuspend the pellet in 1665 µL of Vesicle Isolation Buffer and transfer to a
fresh 2ml Eppi.
SECTION: Day1: Cell lysis and binding to Flag beads
3.3. Incubate on ice for 20 min

SECTION: Day2 Elution of ATG9A vesicles
5. Pellet the beads (in a 5415R centrifuge) at 200 rcf, 3 min and remove the supernatant (>> Sample Unbound).
SECTION: Day2 Elution of ATG9A vesicles
6. Resuspend the beads in 1 mL of Vesicle Isolation Buffer and transfer to a fresh 2ml Eppi (+665 µL Vesicle Isolation Buffer already pre-pipetted in the Eppi).
SECTION: Day2 Elution of ATG9A vesicles
7. Incubate  1 min  rolling at 4 °C or on ice with occasionally inverting the Eppi carefully

SECTION: Day2 Elution of ATG9A vesicles
8. Pellet the beads at 2000 rcf, 3 min  and remove the supernatant
SECTION: Day2 Elution of ATG9A vesicles
9. Resuspend the beads in 1 mLof Vesicle Isolation Buffer and transfer into a fresh 2 ml LowBind tube

SECTION: Day2 Elution of ATG9A vesicles
9.1. Add 665 µL of Elution Base Buffer very slowly to the tube (decreases the sucrose concentration;
now be even more careful than before!)
SECTION: Day2 Elution of ATG9A vesicles
10. Incubate  1 min  rolling at 4 °C or on ice with occasionally inverting the tube carefully

SECTION: Day2 Elution of ATG9A vesicles
11. Pellet the beads at 2000 rcf, 3 min  and remove the supernatant
SECTION: Day2 Elution of ATG9A vesicles
12. Resuspend the beads in 1 mL of Elution Base Buffer and transfer into a fresh 2 ml LowBind tube665 µL Elution Base Buffer already in pre-pipetted in the tube).

SECTION: Day2 Elution of ATG9A vesicles
13. Incubate  1 min  rolling at 4 °C or on ice with occasionally inverting the tube carefully

SECTION: Day2 Elution of ATG9A vesicles
14. Pellet the beads at 2000 rcf, 3 min  and remove the supernatant.

SECTION: Day2 Elution of ATG9A vesicles
15. Resuspend the beads in 666 µL of Elution Base Buffer  and transfer to a fresh 1.5 ml LowBind tube (>> Sample Elution Input).

SECTION: Day2 Elution of ATG9A vesicles
16. Add 16.65 µL of 4 mg FLAG peptide

SECTION: Day2 Elution of ATG9A vesicles
17. Incubate for 165 min at 4°C rolling

SECTION: Day2 Elution of ATG9A vesicles
18. Pellet the beads at 2000 rcf, 3 min and transfer the supernatant to a fresh LowBind tube (>> Sample Elution)
This is your vesicle prep! Handle with care.
SECTION: Day2 Elution of ATG9A vesicles
19. Resuspend the beads again in 666 µL of Elution Base Buffer  (>>Sample Beads after Elution)

SECTION: Preparation of starting material (Hap1 ATG9A-mEGFP-3C-FLAG cell pellets)
1. Grow and expand cells in IMDM medium supplied with 10%
FCS and Pen/Strep until the required number of cells is reached. 
Prep size: For one preparation of vesicles the required amount is
100 x 10^6 to 150 x 10^6 cells which is equivalent to 4 - 5 x 15 cm dishes of confluent
cells.
Detach cells with trypsin, count and aliquot to prep size (see above), wash with cold PBS, spin
down  200 rcf, 4 °C , remove supernatant, flash freeze the cell pellets and keep
at  -80 °C until use.
SECTION: Day1: Cell lysis and binding to Flag beads
2.1. Take 70 µL of FLAG bead slurry per prep.
SECTION: Day1: Cell lysis and binding to Flag beads
3.1.  Thaw cells on ice
SECTION: Day1: Cell lysis and binding to Flag beads
3.8. Transfer the supernatant to a fresh 2 ml Eppi (>> Sample Supernatant)
SECTION: Day1: Cell lysis and binding to Flag beads
3.7. 
Pellet the cell debris and nuclei (200 rcf, 6 min, 4 °C )

SECTION: Day1: Cell lysis and binding to Flag beads
3.6. Repeat the lysis step (push 30 times up and down through a 26 x g needle)

SECTION: Day1: Cell lysis and binding to Flag beads
3.5. Let them chill for 10 min on ice

SECTION: Day1: Cell lysis and binding to Flag beads
3.4. Lyse the cells by pushing them 30 times (up and down) through a 26 x g needle

SECTION: Day1: Cell lysis and binding to Flag beads
4. Add 70 µL of equilibrated FLAG beads (slurry).
   Incubate 10 min at 4 °C (on the roller in the cold room in a 50ml Falcon tube)