Apr 19, 2022

Public workspaceOnepot-seq

This protocol is a draft, published without a DOI.
  • Dongju Shin1,
  • JungWon Choi1,
  • Ji Hyun Lee2,3,
  • Duhee Bang1
  • 1Department of Chemistry, Yonsei University, Seoul, Korea.;
  • 2Department of Clinical Pharmacology and Therapeutics, College of Medicine, Kyung Hee University, Seoul, Korea;
  • 3Department of Biomedical Science and Technology, Kyung Hee University, Seoul, Korea
JungWon Choi
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Protocol Citation: Dongju Shin, JungWon Choi, Ji Hyun Lee, Duhee Bang 2022. Onepot-seq. protocols.io https://protocols.io/view/onepot-seq-b5u3q6yn
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 03, 2022
Last Modified: April 19, 2022
Protocol Integer ID: 59003
Abstract
Onepot-seq protocol follows steps below:
  1. Cells and beads preparation
  2. Scatteration of beads and cells in well
  3. Cell lysis and beads isolation
  4. cDNA synthesis
  5. cDNA Library amplification
  6. 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:
  1. Cells and beads preparation
  2. Scatteration of beads and cells in well
  3. Cell lysis and beads isolation
  4. cDNA synthesis
  5. cDNA Library amplification
  6. 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.
CITATION
Macosko EZ, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M, Tirosh I, Bialas AR, Kamitaki N, Martersteck EM, Trombetta JJ, Weitz DA, Sanes JR, Shalek AK, Regev A, McCarroll SA (2015). Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets.. Cell.
LINK
https://doi.org/10.1016/j.cell.2015.05.002


Materials
- Poly T beads (Chemgene, MACOSKO-2011-10(V+))

- PBSB : PBS supplemented with 0.1% BSA

- incubation buffer : 6% Ficoll PM-400, 20mM EDTA, 0.2M Tris pH 7.5
AB
1ml of Incubation buffer composition
H20 460 μl
20% Ficoll PM-400 (GE healthcare) 300 μl
500mM EDTA (Life Technologies) 40 μl
1M Tris pH 7.5 (Sigma) 200 μl
Total 1000 μl

- Lysis buffer : 1:1 mixture of incubation buffer and 20% Sarkosyl

- 6X SSC

- TE-SDS : TE buffer + 0.5% SDS

- TE-TW : TE buffer + 0.01% Tween-20

- 10mM Tris pH8.0

- DNase free Water (DW)

- Reverse Transcription mix (RT mix)
AB
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

- Exonuclease I mix (Exo I mix)
AB
Exonuclease mix composition
10X ExoⅠbuffer 20 μl
H20 170 μl
ExoⅠ 10 μl
Total 200 μl
- AMPure XP beads
Cells and beads preparation
Cells and beads preparation
Suspend cells in PBS (1,000 cells/μl of concentration is recommended)
Wash Poly T beads 3 times with Amount1 mL PBSB
(PBSB : PBS supplemented with 0.1% BSA)
Suspend beads in incubation buffer (20,000 beads in Amount100 µL incubation buffer)
(incubation buffer : 6% Ficoll PM-400, 20mM EDTA, 0.2M Tris pH 7.5)
Scatteration of beads and cells in well (※ Do experiments in 4C room)
Scatteration of beads and cells in well (※ Do experiments in 4C room)

Add incubation buffer to 12 well plate
Add 1,000 cells (suspended in PBS) to incubation buffer, let the sum of solution volume be Amount900 µL .

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.

Add 20,000 beads (suspended in Amount100 µL incubation buffer)

Gently pipette Amount1 mL incubation buffer to spread the beads and cells evenly

Wait Duration00:15:00 to sink the cells and beads down to the bottom
(Depending on the cell type, it may take more time to sink)
15m
Cell lysis and beads isolation (※ Do experiments in 4C room)
Cell lysis and beads isolation (※ Do experiments in 4C room)
Gently stack upAmount200 µL of lysis buffer on top of incubation buffer
(※ avoid the beads being affected by current flow)

Incubate for Duration00:15:00

15m
Quickly disrupt 1.2ml of solutions with Amount1 mL of 6X SSC

Quickly transfer 2.2ml of solutions toAmount30 mL of 6X SSC (in 50 ml falcon tube)

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)
Pipette 4~5 times to avoid mRNA cross contamination
Centrifuge beads (1000 X g / Duration00:01:00 )
After centrifugation, discard supernatant
1m
Add Amount1 mL of 6X SSC
Transfer beads to 1.5ml tube
Wash beads 3 times with Amount1 mL 6X SSC

Wash beads with ~Amount300 µL of 5X RT buffer

cDNA synthesis (※ similar with Drop-seq protocols)
cDNA synthesis (※ similar with Drop-seq protocols)
2h
2h
Add Amount200 µL Reverse Transcription mix (RT mix)
AB
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

Incubate for Duration00:30:00 at TemperatureRoom temperature using rotator

30m
Incubate forDuration01:30:00 at Temperature42 °C using rotator

1h 30m
Wash the beads once with Amount1 mL TE-SDS

Wash the beads twice with Amount1 mL TE-TW

Wash the beads once with Amount1 mL of Concentration10 millimolar (mM) Tris pH8.0

Add Amount200 µL of Exonuclease I mix (Exo I mix)
AB
Exonuclease mix composition
10X ExoⅠbuffer 20 μl
H20 170 μl
ExoⅠ 10 μl
Total 200 μl

Incubate forDuration00:45:00 at Temperature37 °C using rotator

45m
Wash the beads once with Amount1 mL TE-SDS

Wash the beads twice with Amount1 mL TE-TW

Wash the beads twice with Amount1 mL DW

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

Aliquot Amount10 µL beads containing solutions to 8-strip tube

Add polymerase and PCR primer to each tube
1 reaction of PCR mix composition follows below :
AB
cDNA amplification PCR composition
DW 14.6 μl
beads in DW 10 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)
Run PCR following the protocol below:
AB
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
Collect PCR cDNA library solutions to 1.5ml tube
0.6X Ampure bead purification
elute cDNA library into Amount20 µL DW

NGS library preparation
NGS library preparation
For NGS library prep, follow the Nextera XT DNA Library Preparation Kit (#FC-131-1024) manufacturer's instructions.
Citations
Macosko EZ, Basu A, Satija R, Nemesh J, Shekhar K, Goldman M, Tirosh I, Bialas AR, Kamitaki N, Martersteck EM, Trombetta JJ, Weitz DA, Sanes JR, Shalek AK, Regev A, McCarroll SA. Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets.
https://doi.org/10.1016/j.cell.2015.05.002