TITLE: RT&amp;Tag (Reverse Transcribe &amp; Tagment)
AUTHORS: Nadiya Khyzha

[DESCRIPTION]
Reverse Transcribe & Tagment (RT&Tag) is a method that capitalizes on the ability of the Tn5 transposase to tagment RNA-cDNA hybrids. RT&Tag bypasses immunoprecipitation and instead uses antibodies to tether a protein A-Tn5 transposase fusion protein in situ. By performing localized reverse transcription and tagmentation, one can capture RNAs associated with an epitope of interest within intact nuclei. With RT&Tag, one can generate sequencing libraries using only 100,000 Drosophila cells and require only 8 million sequencing reads per sample.

  


[STEPS]
SECTION: Prepare Concanavalin A beads
2. Mix the Concanavalin A (conA) bead slurry. Transfer 5μl of beads per sample into a 1.5ml eppendorf tube.
Wash with 1ml of Binding Buffer.
Place on magnet and remove the Binding Buffer.
Perform a second wash with 1ml of Binding Buffer.
Resupend in 5μl of Binding Buffer per sample.
Aliquot 5μl of ConA beads into 8-tube PCR strips.

 

SECTION: Prepare nuclei
3. Harvest 4 million S2 cells. 
Centrifuge for 3min at 600g. Remove media. 
Re-suspend in 1ml of Wash Buffer.
Centrifuge for 3min at 600g. Remove Wash Buffer. 
Re-suspend in 500μl of NE1 buffer supplemented with Rnasin Rnase Inhibitor (1U/μl, 1:40 dilution). Immediately place on ice and incubate for 10min.
Centrifuge for 5min at 2500g. Remove NE1 Buffer. 
Re-suspend in 500μl of Wash Buffer supplemented with Rnasin Rnase Inhibitor (1U/μl, 1:40 dilution).
Add 100k nuclei into each of 8-tube PCR strips. Incubate for 10min at room temperature to allow nuclei to bind to beads.

 

SECTION: Reverse Transcription &amp; Tagmentation
8. Prepare reverse transcription master mix by combining per reaction:
       Oligo(dT)-ME-B (12.5mM)         1 μL            dNTP Mix (10mM)         1 μL            5x Reverse Transcription Buffer         4 μL            RNAse Inhibitor (40U/μL)         0.5 μL            Maxima Reverse Transcriptase         0.5 μL            Water         13 μL      
2. Place the PCR strips on a magnet and remove the liquid.
3. Perform 2 sets of washes using 200μl of 300Wash Buffer.
4. Re-suspend beads in 20μl of Reverse Transcription Master Mix while gently vortexing. 
5. Incubate for 2h at 37C in a PCR machine.  
SECTION: Primary antibody binding
4. Prepare Primary Antibody Master Mixes. Use 50μl of Antibody Buffer per reaction. Dilute antibody of interest 1:100 in Antibody Buffer. Supplement with Rnasin Rnase Inhibitor (1U/μl, 1:40 dilution).
Place the PCR strips on a magnet and remove liquid from the nuclei binding step.
Re-suspend beads in 50μl of Primary Antibody Master Mix while gently vortexing. 
Place on a nutator and incubate for 2h at room temperature or overnight at 4°C. 

 

SECTION: Streptavidin conjugated secondary antibody binding
5. Prepare Secondary Antibody Master Mixes. Use 50μl of Wash Buffer per reaction. Dilute streptavidin conjugated secondary antibody 1:100 in Wash Buffer. Supplement with Rnasin Rnase Inhibitor (1U/μl, 1:40 dilution).
Place the PCR strips on a magnet and remove the liquid.
Re-suspend beads in 50μl of Secondary Antibody Master Mix while gently vortexing. 
Place on a nutator and incubate for 45min at room temperature or overnight at 4°C. 

 

 

SECTION: Protein A-Tn5 binding
7. Prepare Protein A-Tn5 Master Mixes. Use 50μl of 300Wash Buffer per reaction. Dilute protein A-Tn5 loaded with ME-A 1:200 in 300Wash Buffer. Supplement with Rnasin Rnase Inhibitor (1U/μl, 1:40 dilution). The protein A-Tn5 dilution may need to be established empirically based on the activity level of purified protein A-Tn5. 
Place the PCR strips on a magnet and remove the liquid.
Perform 2 sets of washes using 200μl of Wash Buffer.
Re-suspend beads in 50μl of Protein A-Tn5 Master Mix while gently vortexing. 
Place on a nutator and incubate for 1h at room temperature. 

SECTION: Tn5 release
9. Place the PCR strips on a magnet and remove the liquid.
Perform a wash using 50μl of Post-tagmentation Wash Buffer.
Add 5μl of SDS Release Buffer to each PCR tube. Don't mix or resuspend by vortexing as the bead solution will be quite sticky at this point.  
Incubate for 1h at 58C in a PCR machine. 
SECTION: Amplifying libraries
10. Set-up PCR reaction master mix by combining per reaction: 

 
2. Add 42 µl of PCR reaction master mix to each sample.
3. Add 2 µl of corresponding 10 µM barcoded i7 primer to each sample. 
4. Perform PCR using the following conditions: 
       Cycle number         Denature         Anneal         Extend         Final            1                             58° C, 5 min                                                    72° C, 5 min                      2         98° C, 30 s                     3–14         98° C, 10 s         60° C, 10 s                  15               72° C, 1 min               16                  4° C, hold      

SECTION: Library clean-up
11. Perform library clean-up using x0.8 AMPure XP beads. To do so mix the 50µl PCR reaction with 40µl of AMPure XP beads in PCR tubes. Vortex at full speed and centrifuge.
Allow to incubate for 5-10min.
Place the PCR tubes on a magnet and allow a few minutes for beads to gather. 
Remove the liquid and perform 2 rounds of washes with 200 µl of 80% ethanol while still on magnet.
Perform a quick centrifuge to gather the remaining ethanol. Place back on magnet and use a 20 µl pipette to remove the residual ethanol.
Re-suspend the beads in 22 µl of 10mM Tris-HCl. Vortex to mix, centrifuge, and allow to incubate for 5-10min.
Place on magnet and transfer the final sample to a clean tube.
SECTION: Tapestation
12. Combine 2 µl of sample with 2µl of sample dye and run on D5000 Tapestation to get library size distribution.
Expected library size should be between 200-700bp. 
  
  3. If large peak around 150bp is observed, perform an additional round of x0.8 AMPure XP bead clean-up.
SECTION: Biotinylated Oligo(dT)-ME-B binding
6. Prepare Biotinylated Oligo(dT)- ME-B Master Mixes. Use 50μl of Wash Buffer per reaction. Dilute biotinylated Oligo(dT)- ME-B (12.5uM) 1:50 in Wash Buffer. Supplement with Rnasin Rnase Inhibitor (1U/μl, 1:40 dilution).
Place the PCR strips on a magnet and remove the liquid.
Perform 2 sets of washes using 200μl of Wash Buffer.
Re-suspend beads in 50μl of Biotinylated Oligo(dT)- ME-B Master while gently vortexing. 
Place on a nutator and incubate for 20min at room temperature. 
SECTION: Prepare Concanavalin A beads
15. 
SECTION: Clean desk and prepare reagents
1. 
 
Prepare all buffers and reagents.