Apr 19, 2022

Public workspaceaa-Onepot-seq

This protocol is a draft, published without a DOI.
  • 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. aa-Onepot-seq. protocols.io https://protocols.io/view/aa-onepot-seq-b7mtrk6n
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: April 15, 2022
Last Modified: April 19, 2022
Protocol Integer ID: 60819
Abstract
aa-Onepot-seq follows protocol below:
  1. Cells and beads preparation and incubation
  2. Transfer and Scatter Cell-bead complexes
  3. Cell lysis and beads isolation
  4. cDNA synthesis
  5. cDNA library amplification
  6. 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:
  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 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:
  1. Cells and beads preparation and incubation
  2. Transfer and Scatter Cell-bead complexes
  3. Cell lysis and beads isolation
  4. cDNA synthesis
  5. cDNA library amplification
  6. NGS library preparation

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


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

- pluriSelect strainer (20μm)

- 10ml syringe
Cells and beads preparation and incubation
Cells and beads preparation and incubation
Prepare 1X106 cells to be stained
Add Amount100 µL of staining buffer to cell pellet and incubate for Duration00:05:00 at Temperature4 °C

5m
Add Amount2 µL ADT Antibody and incubate for Duration00:30:00 at Temperature4 °C

30m
Centrifuge and discard staining buffer
(Determine the centrifugation rcf depending on the cell line)
Wash twice with Amount1 mL PBS

Cell count using hemocytometer
Dilute cells with PBSB (1,000 cells/μl is recommended)
(PBSB : PBS supplemented with 0.1% BSA)
Wash Poly T beads 3 times with Amount1 mL of PBSB

Suspend beads in PBSB (20,000 beads in Amount100 µL PBSB)

Add 20,000 beads to 96 well plate
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)
Transfer and Scatter Cell-bead complexes
Transfer and Scatter Cell-bead complexes

Transfer Amount100 µL of solutions (containing bead-cell complexes) to 20um pluriSelect 20μm strainer
(Check the picture below for strainer and sytinge setting)


Wash 3 times with Amount2 mL PBSB using syringe
(Pulling piston with low pressure to prevent bead-cell complex dissociation)

Invert the strainer and transfer bead-cell complexes to 12 well plate with adding of Amount1 mL incubation buffer
(Incubation buffer : 6% Ficoll PM-400, 20mM EDTA, 0.2M Tris pH 7.5)
Gently pipette Amount1 mL of incubation buffer to spread bead-cell complexes evenly.

Wait Duration00:05:00 to sink the bead-cell complexes down to the bottom

5m
Subsequent process
Subsequent process
The subsequent process is same as Onepot-seq protocol.