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

[DESCRIPTION]
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 preparation

[GUIDELINES]
Onepot-seq is single cell RNA sequencing(scRNA-seq) experiment method.
The main idea of Onepot-seq is to do single cell experiment in continuous medium without compartmentalization.
Therefore, we focus on temperature control and not disturbing the solutions due to transient mRNA localization.

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 preparation

From the 4th step onwards, since it is a process of dealing with beads, it basically follows the Dropseq protocol (Macosko et al., 2015) and there are some modifications.
 



[STEPS]
SECTION: Cells and beads preparation
1. Suspend cells in PBS (1,000 cells/μl of concentration is recommended)
SECTION: Cells and beads preparation
3. Suspend beads in incubation buffer (20,000 beads in 100 µL incubation buffer)
(incubation buffer : 6% Ficoll PM-400, 20mM EDTA, 0.2M Tris pH 7.5)
SECTION: Scatteration of beads and cells in well (※ Do experiments in 4C room)
4. 
Add incubation buffer to 12 well plate
SECTION: Scatteration of beads and cells in well (※ Do experiments in 4C room)
5. Add 1,000 cells (suspended in PBS) to incubation buffer, let the sum of solution volume be 900 µL.

SECTION: Scatteration of beads and cells in well (※ Do experiments in 4C room)
6. Add 20,000 beads (suspended in 100 µL incubation buffer)

SECTION: Scatteration of beads and cells in well (※ Do experiments in 4C room)
7. Gently pipette 1 mL incubation buffer to spread the beads and cells evenly

SECTION: Scatteration of beads and cells in well (※ Do experiments in 4C room)
8. Wait 15 min to sink the cells and beads down to the bottom
(Depending on the cell type, it may take more time to sink)
SECTION: Cell lysis and beads isolation (※ Do experiments in 4C room)
9. Gently stack up200 µL of lysis buffer on top of incubation buffer
(※ avoid the beads being affected by current flow)

SECTION: Cell lysis and beads isolation (※ Do experiments in 4C room)
10. Incubate for 15 min

SECTION: Cell lysis and beads isolation (※ Do experiments in 4C room)
11. Quickly disrupt 1.2ml of solutions with 1 mLof 6X SSC

SECTION: Cell lysis and beads isolation (※ Do experiments in 4C room)
12. Quickly transfer 2.2ml of solutions to30 mL of 6X SSC (in 50 ml falcon tube)

SECTION: Cell lysis and beads isolation (※ Do experiments in 4C room)
13. Pipette 4~5 times to avoid mRNA cross contamination
SECTION: Cell lysis and beads isolation (※ Do experiments in 4C room)
14. Centrifuge beads (1000 X g / 1 min)
After centrifugation, discard supernatant
SECTION: Cell lysis and beads isolation (※ Do experiments in 4C room)
15. Add 1 mL of 6X SSC
SECTION: Cell lysis and beads isolation (※ Do experiments in 4C room)
16. Transfer beads to 1.5ml tube
SECTION: Cell lysis and beads isolation (※ Do experiments in 4C room)
17. Wash beads 3 times with 1 mL 6X SSC

SECTION: Cell lysis and beads isolation (※ Do experiments in 4C room)
18. Wash beads with ~300 µL of 5X RT buffer

SECTION: cDNA synthesis (※ similar with Drop-seq protocols)
19. Add 200 µL Reverse Transcription mix (RT mix)
     RT mix composition        H20      80 μl        Maxima 5X RT buffer      40 μl        20% Ficoll PM-400      40 μl        10mM dNTPs      20 μl        100uM TSO      5 μl        RNase inhibitor      5 μl        Maxima H- RTase      10 μl        Total      200 μl   

SECTION: cDNA synthesis (※ similar with Drop-seq protocols)
20. Incubate for 30 min at Room temperature using rotator

SECTION: cDNA synthesis (※ similar with Drop-seq protocols)
21. Incubate for90 min at 42 °C using rotator

SECTION: cDNA synthesis (※ similar with Drop-seq protocols)
22. Wash the beads once with 1 mL TE-SDS

SECTION: cDNA synthesis (※ similar with Drop-seq protocols)
23. Wash the beads twice with 1 mL TE-TW 

SECTION: cDNA synthesis (※ similar with Drop-seq protocols)
24. Wash the beads once with 1 mL of 10 millimolar (mM) Tris pH8.0

SECTION: cDNA synthesis (※ similar with Drop-seq protocols)
25. Add 200 µL of Exonuclease I mix (Exo I mix)
     Exonuclease mix composition        10X ExoⅠbuffer      20 μl        H20      170 μl        ExoⅠ      10 μl        Total      200 μl   

SECTION: cDNA synthesis (※ similar with Drop-seq protocols)
26. Incubate for45 min at 37 °C using rotator

SECTION: cDNA synthesis (※ similar with Drop-seq protocols)
27. Wash the beads once with 1 mL TE-SDS

SECTION: cDNA synthesis (※ similar with Drop-seq protocols)
28. Wash the beads twice with 1 mL TE-TW

SECTION: cDNA synthesis (※ similar with Drop-seq protocols)
29. Wash the beads twice with 1 mL DW

SECTION: cDNA Library amplification (※ similar with Drop-seq protocols)
30. Resuspend beads with  DW
(2,000-2,500 beads in 10 μL of DW)

SECTION: cDNA Library amplification (※ similar with Drop-seq protocols)
31. Aliquot 10 µLbeads containing solutions to 8-strip tube

SECTION: cDNA Library amplification (※ similar with Drop-seq protocols)
32. Add polymerase and PCR primer to each tube 
1 reaction of PCR mix composition follows below : 
     cDNA amplification PCR composition        H20 + beads      24.6 μl        SMART PCR Primer (100uM)      0.4 μl        2X KAPA      25 μl        Total      50 μl   
(50μL PCR reaction for 100~125 cells / 2,000~2,500 beads)
SECTION: cDNA Library amplification (※ similar with Drop-seq protocols)
33. Run PCR following the protocol below:
 
     PCR running protocol               95C 3min        Denature      98C 20 sec        Annealing      65C 45 sec        Elongation      72C 3 min               4 cycles        Denature      98C 20 sec        Annealing      67C 20 sec        Elongation      72C 3 min               9 cycles        Final extension      72C 5 min        Hold      4C forever   
 
SECTION: cDNA Library amplification (※ similar with Drop-seq protocols)
34. Collect PCR cDNA library solutions to 1.5ml tube
SECTION: cDNA Library amplification (※ similar with Drop-seq protocols)
35. 0.6X Ampure bead purification 
elute cDNA library into 20 µL DW

SECTION: Cells and beads preparation
2. Wash Poly T beads 3 times with 1 mL PBSB
(PBSB : PBS supplemented with 0.1% BSA)
SECTION: NGS library preparation
36. For NGS library prep, follow the Nextera XT DNA Library Preparation Kit (#FC-131-1024) manufacturer's instructions.
SECTION: Cell lysis and beads isolation (※ Do experiments in 4C room)
12.1. If multiple wells or larger area wells are used to increase the number of cells, collect beads corresponding to 5,000 cells in one falcon tube.
(RT mix is effective up to 5,000 cells)
SECTION: Scatteration of beads and cells in well (※ Do experiments in 4C room)
5.1. If you are testing more than 1,000 cells, increase the number of wells or increase the area of ​​the wells according to cells and beads number.