TITLE: FLOUR-seq
AUTHORS: Zhichao Chen

[DESCRIPTION]
We presented three barcoded technologies (BD Rhapsody) that are accessible to most researchers: high-throughput single-cell ONT full-length RNA sequencing (FLOUR-seq). FLOUR-seq combines BD Rhapsody and nanopore sequencing to detect the RNA panorama (including nascent, mature, and degrading RNAs) in cells.The ultralong three barcodes can be discriminated using nanopore technology with 70% debarcoding efficiency and can detect the RNA panorama.

[STEPS]
SECTION: mouse testis cell dissociation
1. The mouse testis were dissociated to single cells refer to Adult mouse testis cell dissociation (on ice).
SECTION: Priming and treating the BD Rhapsody Cartridge
2. Prime and treat the BD Rhapsody Cartridge (Cat. No. 400000847). 

 

SECTION: Loading cells in cartridge
3. Dilute 20000 cells with cold Sample Buffer (Cat. No. 650000062) to 650 μL.
SECTION: Loading cells in cartridge
4. Load cartridge with materials listed using the P1200M pipette:

 

SECTION: Loading cells in cartridge
5. Incubate at room temperature (15°C to 25°C) for 15 minutes. During 15 minute incubation, prepare Cell Capture Beads(Cat. No. 650000089).
SECTION: Preparing, loading and washing Cell Capture Beads
6. Place Cell Capture Bead tube on magnet for 1 minute, and remove storage buffer. 
Remove tube from magnet, and pipet 650 μL cold Sample Buffer (Cat. No. 650000062) into tube.
SECTION: Preparing, loading and washing Cell Capture Beads
7. Set P1200M pipette to Prime/Treatmode. Load cartridge with materials listed using the P1200M pipette: 
   Material to loadVolume (μL)Pipette modeAir700Prime/Treat􏰀 SetP1200MpipettetoBeadLoadmode.􏰀 UseamanualP1000togentlypipet-mixbeadsincoldSampleBuffer (Cat. No. 650000062). Immediately load.Cell Capture Beads630Bead Load

 
 
SECTION: Preparing, loading and washing Cell Capture Beads
8. Incubate the cartridge at room temperature (15°C to 25°C) for 3 minutes.
SECTION: Preparing, loading and washing Cell Capture Beads
9. Place cartridge on the plate shaker plate adapter. Shake the cartridge at room temperature (15°C to 25°C) at 1,000 rpm for 15 seconds.
Return cartridge to Express instrument, and wait 30 seconds.
SECTION: Preparing, loading and washing Cell Capture Beads
10. Set P1200M pipette to Wash mode. Load cartridge with materials listed using the P1200M pipette:
   Material to loadVolume (μL)Pipette modeaAir700WashCold Sample Buffer (Cat. No. 650000062)700WashAir700WashCold Sample Buffer (Cat. No. 650000062)700Wash

 
 
SECTION: Lysing cells
11. Add 75.0 μL 1 M DTT (Cat. No. 650000063) to one 15 mL Lysis Buffer bottle (Cat. No. 650000064). Briefly vortex lysis mix, place on ice.
SECTION: Lysing cells
12. Move the left slider to LYSIS on Express instrument. Set P1200M pipette to Lysis mode. Set P1200M pipette to Lysis mode.
   Material to loadVolume (μL)Pipette modeLysis Buffer with DTT550Lysis
Incubate at room temperature (15°C to 25°C) for 2 minutes.
 
 
SECTION: Retrieving Cell Capture Beads
13. Place the 5 mL LoBind Tube in Express instrument drawer. Ensure P5000M pipette is set to Retrieval mode. Move the front slider to BEADS on Express instrument.
SECTION: Retrieving Cell Capture Beads
14. Move the left slider to RETRIEVAL. Leave Retrieval magnet in down position for 30 seconds. Aspirate 5,000 μL Lysis Buffer with DTT with the P5000M pipette. Press down on P5000M pipette to seal against the gasket. Move the left slider to the middle position (0), andimmediatelyload 4,950 μL Lysis Buffer with DTT.
SECTION: Retrieving Cell Capture Beads
15. Remove pipette from gasket, and purge tip. Move the front slider to OPEN, and place the 5 mL LoBind Tube on large magnet with 15 mL tube adapter (V&P Scientific Cat. No. VP 772FB-1A) for 1 minute.
SECTION: Washing Cell Capture Beads
16. After 1 minute incubation leaving the 5 mL tube containing retrieved Cell Capture Beads on large magnet, remove all but ~1 mL of supernatant without disturbing beads.
SECTION: Washing Cell Capture Beads
17. Remove tube from magnet. Gently pipet-mix beads, and transfer them to a new 1.5 mL LoBind Tube.
SECTION: Washing Cell Capture Beads
18. Place tube on magnet for ≤2 minutes, and remove supernatant. Avoid leaving Lysis Buffer or bubbles in tube. Lysis Buffer might cause the reverse transcription reaction to fail.
SECTION: Washing Cell Capture Beads
19. Remove tube from magnet and pipet 1.0 mL of cold Bead Wash Buffer (Cat. No. 650000065) into tube. Pipet-mix.
SECTION: Washing Cell Capture Beads
20. Place tube on 1.5 mL tube magnet for ≤2 minutes, and remove supernatant.
SECTION: Washing Cell Capture Beads
21. Remove tube from magnet, and pipet 1.0 mL cold Bead Wash Buffer (Cat. No. 650000065) into tube. Pipet-mix, and place on ice.
SECTION: cDNA synthesis and template switching
22. In a new 1.5-mL LoBind tube, pipet the following reagents.
cDNA/Template switching mix

   ComponentFor 1 library (μL)For 1 library with 20% overage (μL)RT Buffer4048dNTP2024RT 0.1 M DTT1012Bead RT/PCR Enhancer1214.4RNase Inhibitor1012Reverse Transcriptase1012Nuclease-free water92110.4Total194232.8

 Gently vortex mix, briefly centrifuge, and place back on ice.
 
SECTION: cDNA synthesis and template switching
23. Place the tube of washed Cell Capture Beads on a 1.5-mL tube magnet for≥2 minutes. Remove the
supernatant and pipet 194 μL of cDNA mix into the beads. Pipet-mix.
SECTION: cDNA synthesis and template switching
24. Incubate the bead suspension on the thermomixer at 1,200 rpm and 42°C for 30 minutes.
SECTION: cDNA synthesis and template switching
27. Resuspend the beads with the saved supernatant in step 25, add 3 μL of template switch oligo (100 uM5'  A AGC AGT GGT ATC AAC GCA GAG TAC rG rG +G 3') , and incubate on the thermomixer for 15 minutes at 1,200 rpm and 42°C. Add 2 μL of 1 M MgCl2 to the reaction mix, and incubate on the thermomixer for another 15 minutes at 1,200 rpm and 42°C.
SECTION: cDNA synthesis and template switching
26. Place tube on 1.5 mL tube magnet for ≤2 minutes, and remove supernatant. Wash the beads twice with 1x RT buffer.
SECTION: cDNA synthesis and template switching
25. Place tube on 1.5 mL tube magnet for ≤2 minutes, and pipet supernatant to a new tube.  Remove tube from magnet, and pipet 200 μL the following reagents into tube.

 

SECTION: cDNA synthesis and template switching
28. Briefly spin the tube with the bead suspension. Place the tube on the magnet for≤1 minute until clear. Remove the supernatant.
SECTION: cDNA synthesis and template switching
29. Remove the tube from the magnet, and pipet 200 μL of cold Bead Resuspension Buffer into the tube. Pipet- mix.
SECTION: Performing PCR1
30. Place the tube beads in Bead Resuspension Buffer on a 1.5-mL magnet for≤1 minute. Remove the supernatant. Suspend the beads with following PCR reaction mix:

 
Universal Oligo: 5’-ACACGACGCTCTTCCGATCT-3'
TSO PCR primer: 5‘-AAGCAGTGGTATCAACGCAGAGTAC-3'
 
SECTION: Performing PCR1
31. Ensuring that the beads are fully resuspended, pipet 25 μL of PCR reaction mix with beads into each of 8 0.2-mL PCR tubes. 
SECTION: Performing PCR1
32. Program the thermal cycler as follows.
 
    StepCyclesTemperatureTimeHot start195°Ca3 minDenaturation11–14b95°C30 sAnnealing60°C3 minExtension72°C1 minFinal extension172°C5 minHold14°C∞

 
 
SECTION: Performing PCR1
33. Pipet-mix and combine the four reactions into a new 1.5-mL LoBind tube. Place the 1.5-mL tube on the magnet for≤1 minute. Pipet the supernatant into the new 1.5-mL LoBind tube without disturbing the beads.
SECTION: Performing PCR1
34. Purify the amplified products by 0.7x Agencourt AMPure XP Beads(Beckman Coulter).
SECTION: Performing PCR2
35. Prepare the PCR mix by combining and mixing the following components:
 

TSO lengthen primer: 5'-CGACATGGCTACGATCCGACAAGCAGTGGTATCAACGCAGAG-3'
Library Forward primer: 5'- AATGATACGGCGACCACCGAGATCTACACTATAGCCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3'
SECTION: Performing PCR2
36. Program the thermal cycler as follows.

    StepCyclesTemperatureTimeHot start195°Ca3 minDenaturation11–14b95°C30 sAnnealing60°C3 minExtension72°C1 minFinal extension172°C5 minHold14°C∞

SECTION: Performing PCR2
37. Purify the amplified products by 0.7x Agencourt AMPure XP Beads(Beckman Coulter).
SECTION: Nanopore sequencing
38. Prepared sequencing using Oxford Nanopore Technologies SQK-LSK109 and sequenced on ONT PromethION platforms.