TITLE: ONT Library Prep for Split-seq cDNA
AUTHORS: Jasmine Sakr

[DESCRIPTION]
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. 

[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.

[STEPS]
SECTION: Preparation
1. Thaw the following reagents:
SECTION: End-prep
4. End-prep Reaction
SECTION: End-prep
5. Clean-Up
SECTION: Preparation
2. Prepare the following:
SECTION: Preparation
3. Set heat block to 65°C.
SECTION: End-prep
4.1. In a 1.5 mL tube, mix the following for each sample:

 

SECTION: End-prep
4.2. Thoroughly mix by pipetting and avoid creating bubbles.
SECTION: End-prep
4.3. Incubate at room temperature for 5 minutes on bench top and 65°C for 5 minutes on thermomixer.
SECTION: End-prep
5.1. Vortex AMPure XP Beads (AXP).
SECTION: End-prep
5.2. Add 60 μL of the resuspended AMPure XP Beads (AXP) to the end-prep reaction(s) and mix by pipetting.
SECTION: End-prep
5.3. Incubate tube(s) on thermomixer at 300 rpm for 5 minutes at room temperature.
SECTION: End-prep
5.4. Spin down the tube(s) and pellet on a magnet.
SECTION: End-prep
5.5. Keep the tube(s) on the magnet and pipette off the supernatant.
SECTION: End-prep
5.6. Keep the tube(s) on the magnet and wash the beads with 200 μL of freshly prepared 80% ethanol without disturbing the pellet.
SECTION: End-prep
5.7. Remove the ethanol using a pipette and discard without disturbing the pellet.
SECTION: End-prep
5.8. Keep the tube(s) on the magnet and wash the beads with 200 μL of freshly prepared 80% ethanol without disturbing the pellet (again).
SECTION: End-prep
5.9. Remove the ethanol using a pipette and discard without disturbing the pellet (again).
SECTION: End-prep
5.10. Allow to dry for ~30 seconds, but do NOT dry the pellet to the point of cracking.
SECTION: End-prep
5.11. Remove tube(s) from magnet and resuspend the pellet in 61 μL Nuclease-free water.
SECTION: End-prep
5.12. Incubate tube(s) for 5 minutes at room temperature.
SECTION: End-prep
5.13. Pellet the beads on a magnet.
SECTION: End-prep
5.14. Remove and retain 60 μL of eluate into a new 1.5 mL tube(s).
SECTION: Adapter Ligation
6. Adapter ligation reaction
SECTION: Adapter Ligation
6.1. In the same 1.5 mL tube, mix in the following order for each sample:


 

SECTION: Preparation
1.1. 
 

SECTION: Preparation
2.1. 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.
SECTION: Preparation
2.2. Prepare fresh 80% ethanol for the wash steps. Each sample will need 400 μL
SECTION: Adapter Ligation
6.2. Thoroughly mix by gently pipetting and avoid creating bubbles.
SECTION: Adapter Ligation
6.3. Incubate tube(s) at room temperature for 10 minutes.
SECTION: Adapter Ligation
7. Clean-up
SECTION: Adapter Ligation
7.1. Vortex AMPure XP Beads (AXP).
SECTION: Adapter Ligation
7.2. Add 40 μL of the resuspended AMPure XP Beads (AXP) to the reaction(s) and mix by pipetting.
SECTION: Adapter Ligation
7.3. Incubate tube(s) on the thermomixer at 300 rpm for 5 minutes at room temperature.
SECTION: Adapter Ligation
7.4. Spin down the tube(s) and pellet on a magnet.
SECTION: Adapter Ligation
7.5. Keep the tube(s) on the magnet and pipette off the supernatant.
SECTION: Adapter Ligation
7.6. Remove tube(s) from magnet and wash the beads by adding 250 μL Short Fragment Buffer (SFB).
SECTION: Adapter Ligation
7.7. Resuspend the beads by pipetting.
SECTION: Adapter Ligation
7.8. Pellet the beads on a magnet and pipette off the supernatant.
SECTION: Adapter Ligation
7.9. Remove tube(s) from magnet and wash the beads by adding 250 μL Short Fragment Buffer (SFB) (again).
SECTION: Adapter Ligation
7.10. Resuspend the beads by pipetting (again).
SECTION: Adapter Ligation
7.11. Pellet the beads on a magnet and pipette off the supernatant (again).
SECTION: Adapter Ligation
7.12. Allow to dry for ~30 seconds, but do NOT dry the pellet to the point of cracking.
SECTION: Adapter Ligation
7.13. Remove tube(s) from magnet and resuspend the pellet in 15 μL Elution Buffer (EB).
SECTION: Adapter Ligation
7.14. Incubate for 5 minutes at room temperature.
SECTION: Adapter Ligation
7.15. Pellet the beads on a magnet.
SECTION: Adapter Ligation
7.16. Remove and retain 15 μL of eluate into a new 1.5 mL tube(s).
SECTION: Adapter Ligation
7.17. Qubit each library and keep on ice until loading or store in -20°C.