TITLE: Barcoded and targeted cDNA  library preparation for Oxford Nanopore Technologies sequencing
AUTHORS: Rosemary A Bamford, Szi Kay Leung, Aaron Jeffries, Jonathan Mill

[DESCRIPTION]
The NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module (NEB) was adapted for the purpose of adding Oxford Nanopore Technologies (ONT) compatible barcodes during reverse transcription of RNA. This is a useful process for multiplexing low input samples for ONT transcriptome library preparation.
An optional step before ONT library preparation is the targeted enrichment of cDNA molecules using IDT hybridisation probes. Here we provide a protocol for this process based on the 'PacBio cDNA capture using IDT xGen Lockdown Probes' protocol. 
Using this approach, up to 100 samples can be barcoded with individual ONT barcodes via reverse transcription. Samples can then be pooled together and a cDNA PCR amplification performed. This allows sufficient material for cDNA enrichment and/or ONT ligation sequencing library preparation.


[BEFORE_START]
Order the bespoke IDT primers and dilute to the correct concentration.

[STEPS]
SECTION: Bespoke barcoding of cDNA
2. Primer annealing for first strand synthesis
Prepare in a PCR tube on ice.  A reaction mix of up to 7 µL  of total RNA (75 ng ) is added to 1 µL  2 millimolar (mM)  TSO-ONT primer, 1 µL  10 millimolar (mM)  dNTPs (NEB) and made up to 9 µL  with nuclease-free H2O. Gently invert a few times and spin briefly. 
 


 

SECTION: cDNA enrichment
10. We performed an adapted version of the 'PacBio cDNA Capture Using IDT xGen Lockdown Probes' protocol (Other Documentation - PacBio) and xGen Lockdown Panel.  

 

SECTION: Library preparation and sequencing
16. Library preparation was performed using ONT’s ligation kit (SQK-LSK114). Follow supplier protocol, which also includes details for sequencing. Recommended library input is 100-200 fmol.
SECTION: Bespoke barcoding of cDNA
3. Incubate the mixture in a thermocycler for 5 min  at 70 °C  with the heated lid at 105 °C , then snap cool on ice for 1 min . 

SECTION: Bespoke barcoding of cDNA
4. Reverse Transcription (RT) and Template Switching
Meanwhile, prepare the reverse transcription mix which consists of 2.5 µL  NEBNext Single Cell RT buffer (lilac); 0.5 µL  NEBNext Template Switching Oligo (lilac); 1 µL  NEBNext Single Cell RT enzyme mix (lilac) and 1.5 µL  nuclease-free water. Mix by gentle inversion and spin briefly. Add 5.5 µL of reverse transcription mix to each sample. Gently invert a few times and spin briefly.

 

SECTION: Bespoke barcoding of cDNA
5. Place the mix in a thermocycler (heated lid at 105 °C ):
 42 °C for 90 min 
 70 °C for 10 min  
hold at 4 °C . 

 

SECTION: Bespoke barcoding of cDNA
6. cDNA amplification by PCR
Prepare the cDNA amplification mix which consists of 25 µL  NEBNext Single
Cell cDNA PCR Master Mix (orange); 1 µL  NEBNext Single Cell cDNA PCR Primer (orange); 0.25 µL  NEBNext Cell Lysis Buffer (10X) (white) and 13.75 µL nuclease-free water. Mix by gentle inversion and spin briefly. Add 40 µL of cDNA amplification mix to each sample. Gently invert a few times and spin briefly.

SECTION: Bespoke barcoding of cDNA
7. PCR cycling conditions:
Heated lid set to 105 °C  
Initial denaturation:
98 °C  for 45 s 

For 14 cycles:
98 °C  for 10 s  
65 °C  for 750 s , 
72 °C  for 3 min , 

Final extension:
72 °C  for 5 min  
hold at 4 °C 

 

 

SECTION: cDNA enrichment
11. Barcoded cDNA is equimolar pooled for a total of 1 µg  cDNA per capture reaction. Prepare in a 0.2 mL  PCR tube.
 


SECTION: Bespoke barcoding of cDNA
8. Cleanup of Amplified cDNA
Bring Promega Pronex beads to Room temperature for 30 min prior to use.
Spin tubes down briefly. 0.85X Promega Pronex beads are added. Gently invert a few times and spin briefly. Incubate at Room temperature  on Hula mixer for 5 min . Briefly spin tubes and place on magnet. Leave beads to settle for 5 min . Perform 2x 80% EtOH washes. Amplified cDNA is eluted in 27 µL 1X TE. 

 

SECTION: Bespoke barcoding of cDNA
9. Dilute sample 1:5 and run an Agilent HS D5000 screentape

 

 

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12. Prepare the wash buffers and Dynabeads M-270 Streptavidin beads provided in the xGen Lockdown Hybridisation and Wash kit and use as per instructions below. The xGen Lockdown Hybridisation and Wash kit is optimal for 3 months at -20 °C and poor yields may occur if the kit has expired.


SECTION: cDNA enrichment
14. Post amplification clean up
Bring AMPure beads to Room temperature for 30 min prior to use.
Spin tubes down briefly. 1X AMPure beads are added. Gently invert a few times and spin briefly. Incubate at Room temperature for 10 min . Briefly spin tubes and place on magnet. Leave beads to settle for 5 min . Perform 2x 80% EtOH washes. Amplified cDNA is eluted in 27 µL  1X TE. 

SECTION: cDNA enrichment
13. Amplification of captured DNA sample

 

 


Split the PCR mix into two tubes, 100 µL each.

Amplify using the following PCR protocol:
 
After amplification, pool the 100 µL reactions.

SECTION: cDNA enrichment
15. Quantification was determined using the Qubit DNA High sensitivity assay
(Invitrogen, UK) (1:5 dilution) and HS D5000 screentape (Agilent, UK) (1:5 dilution).  

 

 

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11.1. 1 µL  of TSO blocker and 1 µL  of polyT blocker (poly(dT)30) oligonucleotides (both at 1 millimolar (mM) ) were added to the pooled cDNA. 

 

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11.2. Dry the mixture using a DNA vacuum concentrator (set temperature to 45 °C ). Do not overdry.

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11.3. Add IDT 2X hybridisation buffer (8.5 µL ), hybridisation buffer enhancer (2.7 µL ) and nuclease free water (1.8 µL ) to the dried-down sample. Replace the lid (do not cover the hole with tape). Mix the reaction by gently tapping the tube, followed by a quick spin. 
 

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11.4. Place the mixture on a 95 °C  thermocycler for 10 min  to denature the cDNA. 

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11.5. After allowing the mixture to cool briefly for 2 min  at Room temperature. 4 µL of xGen Lockdown Panel is added for a total volume of 17 µL. Probes should never be added while at 95 °C .
 

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11.6. After 5 min at Room temperature, briefly spin the tubes and incubate in a thermocycler at 65 °C overnight (lid temperature 100 °C).

SECTION: Bespoke barcoding of cDNA
1. This protocol is adapted from that provided with NEBNext Single Cell/Low Input cDNA Synthesis & Amplification Module (NEB, E6420S). Input and component values have been modified from the original kit protocol.
SECTION: cDNA enrichment
12.1. Prepare wash buffers
 

Preheat the following wash buffers to 65 °C in a heat block or water bath for at least 15 min :
200 µL of 1X wash buffer I
400 µL of 1X stringent wash buffer

Other reagents can be kept at Room temperature 

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12.2. Prepare the capture beads
Allow the Dynabeads M-270 Streptavidin to warm to room temperature for 30 min  prior to use.
Mix the beads thoroughly by vortexing for 15 s 
For a single sample, aliquot 100 µL beads into a 1.5 mL LoBind tube. Scale up volume for multiple samples.
Place the LoBind tube in a magnetic rack. When the supernatant is clear, remove and discard the supernatant being careful not to disturb the beads. Any remaining traces of liquid will be removed with subsequent wash steps. Give it 5 min to settle. The Dynabeads are "filmy" and slow to collect to the side of the tube.
While the LoBind tube is in the magnetic rack, add 200 µL of 1X bead wash buffer. For multiple samples, wash with 200 µL x X samples.
Remove the tube from the magnetic rack and vortex until the beads are in solution.
Quickly spin and place the LoBind tube back in the magnetic rack to collect the beads to the side of the tube. Once clear, remove and discard the liquid.
Repeat steps 5-7 for a total of two washes.
Resuspend by vortexing the beads in 100 µL of 1X bead wash buffer. For multiple samples, scale up accordingly. 
Place the tube in the magnetic rack to collect beads to the side of the tube. Once clear, remove and discard the supernatant. 
The washed beads are now ready to bind the captured DNA. Proceed immediately to the next step. Do not allow the capture beads to dry. Small amounts of residual bead wash buffer will not interfere with binding of DNA to the capture beads.

SECTION: cDNA enrichment
12.3. Bind cDNA to the capture beads
Transfer the 17 µL hybridised probe/sample mixture to the washed capture beads. 
Mix by tapping the tube until the sample is homogenous. 
Incubate in a heat block set to 65 °C for 45 min or transfer the mix to a PCR tube and incubate in a thermocycler (heated lid set to 75 °C ). Hand mix periodically by gently tapping the tube to keep the beads in suspension, every 10 min . 

SECTION: cDNA enrichment
12.4. Wash the captured cDNA
The 1X wash buffer I and 1X stringent wash buffer should have been pre-heated to 65 °C .
After the above incubation, remove the tube from the heat block and add 100 µL  pre-heated 1X wash buffer I.
Mix thoroughly by tapping the tube until the sample is homogenous.
If using a PCR tube, transfer the sample to a 1.5 mL LoBind tube (careful of bubbles).
Place the tube in a magnetic rack to collect the beads to the side of the tube. Remove and discard the liquid once clear.
Remove the tube from the magnetic rack and add 200 µL  of 1X stringent wash buffer heated to 65 °C . Mix by tapping the tube until the sample is homogenous. Work quickly so that the temperature does not drop. 
Incubate at 65 °C for 5 min .
Repeat steps 5-7 for a total of two washes using 1X stringent wash buffer heated to 65 °C .
Place the tubes in the magnetic rack to collect the beads to the side of the tube. Remove and discard the liquid once clear.
Add 200 µL of Room temperature 1X wash buffer I. Hand mix by gently tapping the tube, followed by a quick spin.
Place the tube in the magnetic rack to collect the beads to the side of the tube. Remove and discard the liquid once clear. It will clear quickly but allow 1 min .
Add 200 µL of Room temperature 1X wash buffer II. Hand mix by gently tapping the tube, followed by a quick spin. 
Place the tube in the magnetic rack to collect the beads to the side of the tube. Remove and discard the liquid once clear. Allow 5 min .
Add 200 µL of Room temperature 1X wash buffer III. Hand mix by gently tapping the tube. Quick spin. 
Place the tube in the magnetic rack to collect the beads to the side of the tube. Remove and discard the liquid once clear. Allow 5 min .
Remove the tubes from the magnetic rack and add 50 µL of 1x TE. 
Store the beads plus captured samples at -20 °C or proceed to the next step. It is not necessary to separate the beads from the eluted DNA.