TITLE: Real-time and programmable transcriptome sequencing with PROFIT-seq
AUTHORS: lingling hou, Jinyang Zhang

[DESCRIPTION]
PROgrammable Full-length Isoform Transcriptome sequencing (PROFIT-seq) is a method that enriches target transcripts while maintaining unbiased quantification of the whole transcriptome. PROFIT-seq employs combinatorial reverse transcription to capture polyadenylated, non-polyadenylated, and circular RNAs, coupled with nanopore adaptive sampling that selectively enriches target transcripts during sequencing.

[BEFORE_START]
All experimental procedures including reagent preparation should be performed under RNase- and DNase-free conditions. Bleach the workbench with RNaseZap before starting the experiment. Change gloves frequently to avoid RNase contamination.

Primer:
- Order 100 nmol of DNA oligo, PAGE purification.
- Dissolve the primer powder in nuclease-free water to a final concentration of 50 µM.
- The dissolved primers can be stored at -20°C for months.
 
- RT.dT.ds(1.4uM) can be obtained by annealing RT.dT (50 µM) and RT.ds.rv (50 µM) 1:1 at 1.4 μM in anealing buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl). 
- RT.N6.ds(1.4uM) can be obtained by annealing RT.N6 (50 µM) and RT.ds.rv (50 µM) 1:1 at 1.4 μM in anealing buffer (10 mM Tris-HCl pH 7.5, 50 mM NaCl). 
 
DNA Splint preparation:
- The DNA splint was generated by primer extension of four oligos: RT.linker RT.linker, TSO Linker.R (50 µM), TSO and Linker.R.

[GUIDELINES]
- Handle RNA and enzymes on ice whenever possible.
- All reagents must be kept nuclease-free.
- Use only molecular grade nuclease-free water.
- Perform the incubation steps in the thermal cycler.
- When performing multiple reactions, use a master mix containing 10% excess.
- If not mentioned otherwise, use a 1:1 ratio of beads to sample to select nucleic acids >150-200 nt in clean-ups step using magnetic beads.

[STEPS]
SECTION: Ribosomal RNA depletion
0.1. Remove ribosomal RNA from extracted RNA samples:
SECTION: 4. Nanopore sequencing
14. Install and configuration MinKNOW
SECTION: Reverse transcription
1.7. Add 0.5 µLof 1.4 micromolar (µM)RT.N6.ds oligos to the mixture.

SECTION: Reverse transcription
1.10. Prepare the following mix containing the components listed below in a 0.2 mL RNase/DNase-free PCR tube:

 
 
SECTION: Reverse transcription
1. Combinatorial reverse transcription using RT.dT.ds / RT.N6.ds / RT.N6.ss primers:
Capture polyadenylated / non-polyadenylated and circular transcriptome using combinatorial RT primers
SECTION: Reverse transcription
1.13. Add 0.5 µL of RT.N6.ss (50 micromolar (µM)) to the reaction

SECTION: Reverse transcription
1.12. Place the tube in a thermal cycler and Incubate at42 °Cfor 60 min.

SECTION: Reverse transcription
1.9. Incubated at 25 °Cfor 10 min.
SECTION: Reverse transcription
1.15. Place the tube in the thermal cycler and incubate at 25 °C for 10 min, 42 °C for 60 min, and then70 °Cfor 10 min, and bring the sample to 4 °C before proceeding to the next step.

SECTION: Reverse transcription
3. Pre-circularization amplification:
SECTION: Reverse transcription
3.1. Add the following components to a 0.2 mL PCR tube on ice:
 
 

SECTION: Reverse transcription
4.1. Purification the amplified cDNA with AMPure XP beads as described before using a 0.8:1 ratio (40 µL beads). Elute cDNA in 10 µL. 

SECTION: Circularization &amp; rolling circle amplification
5.1. Set up the following reaction mix on ice:
 
 
 
SECTION: Circularization &amp; rolling circle amplification
5.2. Incubate at 50 °C for 60 min.

SECTION: Reverse transcription
3.3. Transfer the PCR tubes to a thermocycler and begin the program below:

 
 
SECTION: Circularization &amp; rolling circle amplification
5. Splint-based circularization:
The cDNA libraries were constructed using the 10X R2C2 protocol.
 

SECTION: Circularization &amp; rolling circle amplification
8. Rolling circle amplification (RCA):
SECTION: Circularization &amp; rolling circle amplification
8.1. Set up the following reaction mix on ice:
 
 

SECTION: Circularization &amp; rolling circle amplification
8.3. Incubated at 30 °C60 min, then 65 °Cfor 20 min.

SECTION: Circularization &amp; rolling circle amplification
9.1. Mix each two tubes of sample into one tube.
SECTION: Circularization &amp; rolling circle amplification
10. Debranching of the RCA products:
SECTION: Circularization &amp; rolling circle amplification
10.3. Place the tubes on a magnet, and aspirate the supernatant into a new tube.
SECTION: Circularization &amp; rolling circle amplification
10.1. Add the T7 Endonuclease I (NEB: M0302L) reaction mix to the beads above to debranch and elute the RCA product:

 
 
SECTION: Circularization &amp; rolling circle amplification
12. Size selection of the RCA products
SECTION: 4. Nanopore sequencing
14.1. Install MinKNOW following the instructions from nanopore community
SECTION: 4. Nanopore sequencing
15. Install PROFIT-seq
SECTION: 4. Nanopore sequencing
14.2. Backup the original sequencing configuration file:

 

SECTION: 4. Nanopore sequencing
14.3. Edit the following line to change reads break intervals:

 

SECTION: 4. Nanopore sequencing
15.1. Install anaconda3

 

SECTION: 4. Nanopore sequencing
15.2. Create virtual environment for PROFIT-seq
 

SECTION: 4. Nanopore sequencing
15.3. Install PROFIT-seq

 

PROFIT-seq is based on ReadUntil API
 

 

SECTION: 4. Nanopore sequencing
15.4. Install other dependencies:

 


 

 

 

 

 

SECTION: 4. Nanopore sequencing
16. Prepare reference genome and annotation:

Prepare the minimap2 index of reference genome

 

SECTION: 4. Nanopore sequencing
17. Connect MinION Mk1B and insert the FLO-MIN106D flow cell correctly:

Open the MinKNOW software to make sure flow cell have been successfully recognized.
SECTION: 4. Nanopore sequencing
18. Run PROFIT-seq
SECTION: 4. Nanopore sequencing
18.1.  Switch to minknow user to start PROFIT-seq process:
 

 

 

 

SECTION: 4. Nanopore sequencing
18.2. If everything works fine, the prompt of url for dashboard will appear on your screen. For example:

 

SECTION: 4. Nanopore sequencing
18.5. Start sequencing protocol in MinKNOW.
SECTION: 5. Data analysis
19. Raw data basecalling:
Re-basecall the sequenced reads with hac model

 


SECTION: 5. Data analysis
20. Channel demultiplex (optional):
If various adaptive sampling jobs are assigned to different channels, use scripts/step1_demultiplex.py to demultiplex reads from different sequencing channels.

 

 


SECTION: 5. Data analysis
21. Adapter trimming & consensus calling:
SECTION: 5. Data analysis
22. Isoform assembly & quantification:

Perform downstream analysis using scripts/step3_analysis.py:

 

 


SECTION: 5. Data analysis
21.1. Trim sequencing barcodes using porechop:

 

SECTION: 5. Data analysis
21.2. Get consensus reads using scripts/step2_consensus.py

 

 


SECTION: 4. Nanopore sequencing
18.3. Access the control panel from the given URL (in this case: http://10.4.0.44:55280)


 

SECTION: 4. Nanopore sequencing
18.4. Configure the adaptive sampling jobs using the "Adaptive Sampling Jobs" panel or upload jobs with the TOML format.

 

SECTION: 4. Nanopore sequencing
18.6. Click 'Run unblock' in PROFIT-seq control panel to start pore manipulation.
SECTION: Ribosomal RNA depletion
0.3. Equilibrate the RNAClean XP beads for at least 30 minat Room temperature , and vortex thoroughly to resuspend the beads.

SECTION: Ribosomal RNA depletion
0.4. Remove ribosomal RNA (rRNA) using an RNase H-based commercial kit – RiboErase kit (human/mouse/rat) (KAPA Biosystems, KK8481) according to the manufacturer’s user guide, and elute RNA in15 µLnuclease-free water.

SECTION: Ribosomal RNA depletion
0.5. Aspirate 2 µL RNA for quantification with a Qubit fluorometer to assess the recovery after rRNA
depletion.
SECTION: Ribosomal RNA depletion
0.6. Aspirate another1 µL RNA for detecting whether mRNA is degraded or not with 5200 Fragment
Analyzer systems (Agilent).

SECTION: Ribosomal RNA depletion
0.2. Use 1 µg total RNA (RNA Quality Number (RQN) ≥ 7.0) as starting material. 
SECTION: Reverse transcription
1.2. Mix by pipetting and spin down brieﬂy in a microcentrifuge.
SECTION: Reverse transcription
1.3. Incubate in a hot-lid thermal cycler at 65 °Cfor 5 min, then place on ice immediately.

SECTION: Reverse transcription
1.4. Prepare the following reagents in a 0.2 mL RNase/DNase-free PCR tube:
 
 

SECTION: Reverse transcription
1.5. Mix by pipetting and spin down brieﬂy in a microcentrifuge.
SECTION: Reverse transcription
1.6. Incubate at 25 °C for 10 min.

SECTION: Reverse transcription
1.1. Adjust the volume of1 µgRNA to 10 µL with nuclease-free water. 
SECTION: Reverse transcription
1.8. Mix by pipetting, and spin the tubes brieﬂy in a microcentrifuge.
SECTION: Reverse transcription
1.11. Mix by pipetting, and spin the tubes brieﬂy in a microcentrifuge.
SECTION: Reverse transcription
1.14. Mix by pipetting, and spin the tubes brieﬂy in a microcentrifuge.
SECTION: Reverse transcription
2. Clean-up:
SECTION: Reverse transcription
2.1. Vortex thoroughly to resuspend the AMPure XP beads before use.
SECTION: Reverse transcription
2.2. Add 40 µLAMPure XP (Beckman, A63880) beads to the 40 µLRNA sample from step 2.15 and mix the sample well by pipetting up and down and spinning down briefly.

SECTION: Reverse transcription
2.3. Incubate at Room temperature for 5 min to bind RNA to the beads.

SECTION: Reverse transcription
2.4. Place the 0.2 mLtube on the magnetic stand until the solution is clear (~2 min). Keep the tube on the magnetic stand. Remove supernatant carefully, taking care not to disturb the beads.

SECTION: Reverse transcription
2.5. Add 200 µLof freshly prepared 75 % (v/v)ethanol to the tube. Wait for 30 s, then discard the entire supernatant.

▲ CRITICAL STEP Beads should always be kept on the magnetic stand while washing with ethanol, and should not be resuspended.
SECTION: Reverse transcription
2.6. Wash beads once more with 75 % (v/v) ethanol by repeating step 3.5  .

SECTION: Reverse transcription
2.7. Quickly spin the tubes in a minicentrifuge and remove all residual liquid.
SECTION: Reverse transcription
2.8. Air-dry the beads on the magnetic rack for 30 s. 

▲CRITICAL STEP Beads should not be overdried during the process, because cracked beads will decrease the RNA recovery.
SECTION: Reverse transcription
2.9. Resuspend beads with 10 µLnuclease-free water.

SECTION: Reverse transcription
2.10. Incubate at room temperature for 2 min to elute RNA from the beads.
SECTION: Reverse transcription
2.11. Put the tube back on the magnetic rack for1 minto pellet the beads.

SECTION: Reverse transcription
2.12. Transfer 8 µLsupernatant to a new0.2 mLtube. Take care not to disturb the bead pellet. The purified cDNA can be stored at -20 °C for several weeks.

SECTION: Reverse transcription
3.2. Mix by pipetting, and spin the tubes brieﬂy in a microcentrifuge.
SECTION: Reverse transcription
4. Clean-up
SECTION: Reverse transcription
4.2. The cDNA can be stored at -20 °C for several weeks.

SECTION: Circularization &amp; rolling circle amplification
6.1. Add 1 µL Exonuclease I (20 U / μl) and 0.3 µL Exonuclease III (100 U / μl) to digest non-circular DNA.

SECTION: Circularization &amp; rolling circle amplification
6.2. Mix by pipetting, and spin the tubes brieﬂy in a microcentrifuge.
SECTION: Circularization &amp; rolling circle amplification
6.3. Incubate at 37 °Cfor 60 min.

SECTION: Circularization &amp; rolling circle amplification
6. Digestion of non-circularized products:
SECTION: Circularization &amp; rolling circle amplification
7. Clean-up:
SECTION: Circularization &amp; rolling circle amplification
7.1. Purification cDNA with AMPure XP beads as described before. 
SECTION: Circularization &amp; rolling circle amplification
7.2. Elute in 10 µL of elution buffer (10 millimolar (mM) Tris, pH 8.0).

SECTION: Circularization &amp; rolling circle amplification
7.3. The cDNA library can be stored at -20 °C for several weeks.

SECTION: Circularization &amp; rolling circle amplification
8.2. Divide the mixture into 4 tubes of 50 µLreaction.
SECTION: Circularization &amp; rolling circle amplification
9. Clean-up:
SECTION: Circularization &amp; rolling circle amplification
9.2. Purification cDNA with AMPure XP beads as described before using a 0.5:1 ratio (50 µl beads).
SECTION: Circularization &amp; rolling circle amplification
9.3. Elute in T7 Endonuclease I reaction mix below.
SECTION: Circularization &amp; rolling circle amplification
10.2. Incubate on a thermal shaker at 37 °C for 30 min under constant agitation at 1800 rpm.

SECTION: Circularization &amp; rolling circle amplification
11. Clean-up
SECTION: Circularization &amp; rolling circle amplification
11.1. Purification the DNA in supernatant with AMPure XP beads as described before using a 0.5:1 ratio (30 µL beads). 

SECTION: Circularization &amp; rolling circle amplification
11.2. Elute in 15 µLof elution buffer (10 millimolar (mM) Tris, pH 8.0).

SECTION: Circularization &amp; rolling circle amplification
11.3. The debranched RCA products can be stored at -20 °Cfor several weeks.

SECTION: Circularization &amp; rolling circle amplification
12.1. Warm the agarose gel cassettes and reagents to room temperature, vortex and spin the
loading solution briefly, and flick and briefly spin the S1 marker. 
SECTION: Circularization &amp; rolling circle amplification
12.2. Add 40 µLrunning buffer to the elution wells following the general guidelines for preparing samples and cassettes as described in the BluePippin User Guide. Samples can be vortexed briefly to properly mix them with the loading solution.

SECTION: Circularization &amp; rolling circle amplification
12.3. Set the program to recover DNA fragments larger than 7 kb using the BluePippin (Sage Science)
and BLF7510 cassette with the ‘0.75DF 1-10 kb Marker S1-Improved Recovery’ cassette definition.
SECTION: Circularization &amp; rolling circle amplification
13. Nanopore sequencing library construction
SECTION: Circularization &amp; rolling circle amplification
13.1. Prepare the cDNA libraries following the Oxford Nanopore protocol “Ligation sequencing amplicons - native barcoding (SQK-LSK109 with EXP-NBD104 and EXP-NBD114)”. The specific steps include end-prep, native barcode ligation and nanopore adapter ligation, and flow cell priming and loading. The updated barcoding kit (SQK-LSK114) can replace SQK-LSK109 for this experiment. 


 

SECTION: 5. Data analysis
23. Output files: