TITLE: aa-Onepot-seq
AUTHORS: Dongju  Shin, JungWon  Choi, Ji  Hyun Lee, Duhee  Bang

[DESCRIPTION]
aa-Onepot-seq follows protocol below:
Cells and beads preparation and incubation
Transfer and Scatter Cell-bead complexes
Cell lysis and beads isolation
cDNA synthesis 
cDNA library amplification
NGS library preparation

[GUIDELINES]
This protocol is modified based on Onepot-seq protocol.
https://www.protocols.io/view/onepot-seq-b5u3q6yn

Onepot-seq protocol follows steps below:
Cells and beads preparation
Scatteration of beads and cells in well
Cell lysis and beads isolation
cDNA synthesis 
cDNA library amplification
NGS library preparation

aa-Onepot-Seq protocol requires additional steps in step1 and 2 from Onepot-Seq protocol
The subsequent process is same as Onepot-seq protocol.

aa-Onepot-seq follows protocol below:
Cells and beads preparation and incubation
Transfer and Scatter Cell-bead complexes
Cell lysis and beads isolation
cDNA synthesis 
cDNA library amplification
NGS library preparation

Therefore, this protocol describes only the parts added to step 1 and 2.



[STEPS]
SECTION: Cells and beads preparation and incubation
1. Prepare 1X106 cells to be stained
SECTION: Cells and beads preparation and incubation
2. Add 100 µL  of staining buffer to cell pellet and incubate for  5 minat 4 °C 

SECTION: Cells and beads preparation and incubation
3. Add 2 µLADT Antibody and incubate for 30 min at 4 °C

SECTION: Cells and beads preparation and incubation
4. Centrifuge and discard staining buffer
(Determine the centrifugation rcf depending on the cell line)
SECTION: Cells and beads preparation and incubation
5. Wash twice with 1 mL PBS

SECTION: Cells and beads preparation and incubation
6. Cell count using hemocytometer
SECTION: Cells and beads preparation and incubation
7. Dilute cells with PBSB (1,000 cells/μl is recommended)
(PBSB : PBS supplemented with 0.1% BSA)
SECTION: Cells and beads preparation and incubation
8. Wash Poly T beads 3 times with 1 mL of PBSB

SECTION: Cells and beads preparation and incubation
9. Suspend beads in PBSB (20,000 beads in 100 µL PBSB)

SECTION: Cells and beads preparation and incubation
10. Add 20,000 beads to 96 well plate
SECTION: Cells and beads preparation and incubation
11. Add 10,000 cells to beads and incubate for 1h at 4C
※ In PBMC experiment, considering the ratio of each cell types of PBMC
(Since antibody efficiencies vary by cell type, the number of cells should be adjusted for each experiment)
SECTION: Transfer and Scatter Cell-bead complexes
12. 
Transfer 100 µL of solutions (containing bead-cell complexes) to 20um pluriSelect 20μm strainer
(Check the picture below for strainer and sytinge setting)
  

SECTION: Transfer and Scatter Cell-bead complexes
13. Wash 3 times with 2 mL PBSB using syringe 
(Pulling piston with low pressure to prevent bead-cell complex dissociation)
 

SECTION: Transfer and Scatter Cell-bead complexes
14. Invert the strainer and transfer bead-cell complexes to 12 well plate with adding of 1 mL incubation buffer
(Incubation buffer : 6% Ficoll PM-400, 20mM EDTA, 0.2M Tris pH 7.5)
SECTION: Transfer and Scatter Cell-bead complexes
15. Gently pipette 1 mL of incubation buffer to spread bead-cell complexes evenly.

SECTION: Transfer and Scatter Cell-bead complexes
16. Wait 5 min to sink the bead-cell complexes down to the bottom

SECTION: Subsequent process
17. The subsequent process is same as Onepot-seq protocol.