Apr 22, 2024
ONT Library Prep for Split-seq cDNA
This protocol is a draft, published without a DOI.
- 1UCI
Protocol Citation: Jasmine Sakr 2024. ONT Library Prep for Split-seq cDNA. protocols.io https://protocols.io/view/ont-library-prep-for-split-seq-cdna-c4spywdn
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: November 08, 2023
Last Modified: April 22, 2024
Protocol Integer ID: 90671
Keywords: Nanopore, ONT, Parse Bio, Split-seq, Long read snRNA-seq, Long read, LR-Split-seq
Abstract
This protocol outlines the preparation of single-cell barcoded cDNA for Oxford Nanopore long-read sequencing. The input cDNA was initially barcoded using the Parse Biosciences Evercode platform for single-nucleus or single-cell RNA-seq (refer to the protocol “Evercode WT v2.2.1” at dx.doi.org/10.17504/protocols.io.eq2lyj9relx9/v1 or “Evercode WT Mega v2.2.1" at dx.doi.org/10.17504/protocols.io.8epv5xxrng1b/v1).
Since we typically perform single-nucleus RNA-seq rather than single-cell RNA-seq, we enrich the cDNA for exome-containing fragments to reduce the number of intronic reads. This is achieved using Twist Biosciences exome panels and a modified version of the Parse Biosciences Gene Capture protocol (refer to the protocol “cDNA Exome Capture v1.0.1” at dx.doi.org/10.17504/protocols.io.36wgq3b83lk5/v1).
The resulting product of this protocol is full-length, barcoded cDNA, excluding intron-only fragments, with Nanopore adapters added to both ends, creating a final library ready for sequencing. The first part of the protocol after setup, End Prep, involves using NEBNext reagents to repair the cDNA ends, resulting in 5′ phosphorylated, 3′ dA-tailed ends, followed by a bead-based cleanup. The next part, Adapter Ligation, adds Nanopore adapters to the end-prepped cDNA, followed by a bead-based cleanup and final elution.
Please see the attachment for the original protocol.
Attachments
Materials
REAGENTS
| Item | Supplier | Part Number | Notes | |
| Ligation Sequencing Kit V14 | Oxford Nanopore Technologies | SQK-LSK114 | The Long Fragment Buffer (LFB) will NOT be used because it selects for fragments that are 3kb and longer. | |
| NEBNext Ultra II End repair/dA-tailing Module | New England BioLabs | E7546 | ||
| NEBNext Quick Ligation Module | New England BioLabs | E6056 | NEBNext Quick Ligation Reaction Buffer will NOT be used. | |
| AMPure® XP Reagent | Beckman Coulter | A63880 (5 mL) A63881 (60 mL) | ||
| Nuclease-Free Water | Sigma-Aldrich | W4502 | Or equivalent nuclease-free water. | |
| Ethyl Alcohol, Pure | Sigma-Aldrich | 459844 | Or equivalent 100% non-denatured ethanol. |
EQUIPMENT
| A | B | C | D | |
| Thermomixer | Various Suppliers | Varies | Compatible with 1.5 mL tubes. | |
| Microcentrifuge | Various Suppliers | Varies | Compatible with 1.5 mL tubes. | |
| Single Channel Pipettes: P2, P20, P200, P1000 | Various Suppliers | Varies | ||
| Vortex-Genie 2 | Scientific Industries | SI-0236 | Or an equivalent vortex mixer. | |
| 6-Tube Magnetic Separation Rack | New England Biolabs | S1506S | Or an equivalent magnetic rack for 1.5 mL tubes. | |
| Qubit Flex Fluorometer | Thermo Fisher Scientific | Q33327 | Or an equivalent fluorometer. |
SUPPLIES
| A | B | C | D | |
| Pipette Tips TR LTS 10 μL, 20 μL, 200 μL, 1,000 μL | Rainin | 17014961 17014963 17014967 | Or appropriate sterile, DNA low-binding, and filtered pipette tips. We do not recommend using wide bore tips. | |
| DNA LoBind® Tubes, 1.5 mL, Snap Cap | Eppendorf | 022431021 | Or equivalent DNA low-binding, nuclease-free 1.5 mL tubes. | |
| Qubit dsDNA HS (High Sensitivity) Assay Kit | Thermo Fisher Scientific | Q33230 (100 assays) Q33231 (500 assays) | Or equivalent fluorescent DNA dye based quantification kit. | |
| Ice bucket | Various Suppliers | Varies |
Before start
Input:
This protocol needs 100-200 fmol of full-length cDNA. We recommend using 200 fmol to get the highest yield. This concentration could be calculated with average basepair length and weight using an online calculator. cDNA fragments with average basepair length between 1-1.5 kb will need between 130-200ng of total cDNA as input. Library yields will range between 3-6.5 ng/μL.
Preparation
Preparation
Thaw the following reagents:
| Item | Location | Handling and Storage | |
| DNA Control Sample (DCS) | Ligation Sequencing Kit V14 (-20°C) | Thaw on ice and mix by pipetting. | |
| Ligation Adapter (LA) | Ligation Sequencing Kit V14 (-20°C) | Thaw on ice and mix by pipetting, do NOT vortex. | |
| Ligation Buffer (LNB) | Ligation Sequencing Kit V14 (-20°C) | Due to viscosity, vortexing this buffer is ineffective. Spin down and mix by pipetting. | |
| Short Fragment Buffer (SFB) | Ligation Sequencing Kit V14 (-20°C) | Thaw on ice and mix well by vortexing. | |
| Elution Buffer (EB) | Ligation Sequencing Kit V14 (-20°C) | Thaw on ice and mix well by vortexing. | |
| Ultra II End-Prep Enzyme Mix | NEBNext Ultra II End repair/dA-tailing Module (-20°C) | Thaw on ice and mix by pipetting, do NOT vortex. | |
| Ultra II End-Prep Reaction Buffer | NEBNext Ultra II End repair/dA-tailing Module (-20°C) | Thaw on ice and mix well by vortexing. Make sure there is no precipitate. | |
| Ultra II End Prep Enzyme Mix | NEBNext Quick Ligation Module (-20°C) | Thaw on ice and mix by pipetting, do NOT vortex. | |
| AMPure XP Beads (AXP) | 4°C | Equilibrate to room temperature. |
Prepare the following:
For each sample: Adjust 100-200 fmol of cDNA volume to 49 μL with Nuclease-free water in a 1.5 mL tube and mix thoroughly by pipetting up and down.
Prepare fresh 80% ethanol for the wash steps. Each sample will need 400 μL
Set heat block to 65°C.
End-prep
End-prep
End-prep Reaction
In a 1.5 mL tube, mix the following for each sample:
| Item | Volume (μL) | |
| DNA CS | 1 | |
| cDNA sample | 49 | |
| Ultra II End-prep Reaction Buffer | 7 | |
| Ultra II End-prep Enzyme Mix | 3 |
Thoroughly mix by pipetting and avoid creating bubbles.
Incubate at room temperature for 5 minutes on bench top and 65°C for 5 minutes on thermomixer.
Clean-Up
Vortex AMPure XP Beads (AXP).
Add 60 μL of the resuspended AMPure XP Beads (AXP) to the end-prep reaction(s) and mix by pipetting.
Incubate tube(s) on thermomixer at 300 rpm for 5 minutes at room temperature.
Spin down the tube(s) and pellet on a magnet.
Keep the tube(s) on the magnet and pipette off the supernatant.
Keep the tube(s) on the magnet and wash the beads with 200 μL of freshly prepared 80% ethanol without disturbing the pellet.
Remove the ethanol using a pipette and discard without disturbing the pellet.
Keep the tube(s) on the magnet and wash the beads with 200 μL of freshly prepared 80% ethanol without disturbing the pellet (again).
Remove the ethanol using a pipette and discard without disturbing the pellet (again).
Allow to dry for ~30 seconds, but do NOT dry the pellet to the point of cracking.
Remove tube(s) from magnet and resuspend the pellet in 61 μL Nuclease-free water.
Incubate tube(s) for 5 minutes at room temperature.
Pellet the beads on a magnet.
Remove and retain 60 μL of eluate into a new 1.5 mL tube(s).
Adapter Ligation
Adapter Ligation
Adapter ligation reaction
In the same 1.5 mL tube, mix in the following order for each sample:
| Item | Volume (μL) | |
| cDNA sample (from the previous step) | 60 | |
| Ligation Adapter (LA) | 5 | |
| Ligation Buffer (LNB) | 25 | |
| NEBNext Quick T4 DNA Ligase | 10 |
Thoroughly mix by gently pipetting and avoid creating bubbles.
Incubate tube(s) at room temperature for 10 minutes.
Clean-up
Vortex AMPure XP Beads (AXP).
Add 40 μL of the resuspended AMPure XP Beads (AXP) to the reaction(s) and mix by pipetting.
Incubate tube(s) on the thermomixer at 300 rpm for 5 minutes at room temperature.
Spin down the tube(s) and pellet on a magnet.
Keep the tube(s) on the magnet and pipette off the supernatant.
Remove tube(s) from magnet and wash the beads by adding 250 μL Short Fragment Buffer (SFB).
Resuspend the beads by pipetting.
Pellet the beads on a magnet and pipette off the supernatant.
Remove tube(s) from magnet and wash the beads by adding 250 μL Short Fragment Buffer (SFB) (again).
Resuspend the beads by pipetting (again).
Pellet the beads on a magnet and pipette off the supernatant (again).
Allow to dry for ~30 seconds, but do NOT dry the pellet to the point of cracking.
Remove tube(s) from magnet and resuspend the pellet in 15 μL Elution Buffer (EB).
Incubate for 5 minutes at room temperature.
Pellet the beads on a magnet.
Remove and retain 15 μL of eluate into a new 1.5 mL tube(s).
Qubit each library and keep on ice until loading or store in -20°C.
Protocol references
Please see the attachment for the original Oxford Nanopore Technologies protocol.