# Goal/Experiment:
The goal of this experiment is to perform a 3-level sci RNA-Seq (single-cell combinatorial indexing RNA sequencing) with the addition of Fluorescence Activated Cell Sorting (FACS) to decrease background noise during the library preparation stage.
## 3-level sci RNA-Seq with FACS
**David Fraser Read¹, Cole Trapnell¹**
¹University of Washington, Dept. of Genome Sciences
**DOI:** [dx.doi.org/10.17504/protocols.io.buxdnxi6](https://dx.doi.org/10.17504/protocols.io.buxdnxi6)
### Abstract
This protocol is a variant of "3 level sci RNA-Seq" which includes a FACS sorting step before the PCR stage. This notable addition helps to decrease background in the library preparation.
### Guidelines
This protocol is an adaptation and includes:
- Addition of FACS sorting to reduce background noise.
- Omission of the USER enzyme reaction step.
- Modified reverse transcription temperature ramp to enhance the number of unique molecular identifiers (UMIs) recovered per nucleus.
### Materials
#### Supplies
- **Nuclease-free water** (Ambion, AM 9937)
- **Snap Cap FACS Tube** (Corning, 08-771-23)
- **SUPERase In RNase Inhibitor 20 U/μL** (Thermo Fisher Scientific, AM2696)
- **BSA 20 mg/mL** (NEB, B9000S)
- **1M Tris-HCl (pH 7.4)** (Thermo Fisher Scientific, AM9759)
- **5M NaCl** (Thermo Fisher Scientific, AM9759)
- **1M MgCl2** (Thermo Fisher Scientific, AM9530G)
- **Triton X-100 for molecular biology** (Sigma Aldrich, 93443-100ML)
- **10mM dNTP** (Thermo Fisher Scientific, R0192)
- Indexed oligo-dT primers (100uM, 5'/5Phos/CAGACGNNNNNNNNNNT10bp barcode)
- **Superscript IV RNase Inhibitor** (Invitrogen, 10777019)
- **Quick ligation kit** (NEB, M2200L)
- **Elution buffer** (Qiagen, 19086)
- **NEBNext Ultra II Non-Directional RNA Second Strand Synthesis Module** (NEB, E7550S)
- **DNA binding buffer** (Zymo Research, D4004-1-L)
- **AMPure XP beads** (Beckman Coulter, A63882)
- **Ethanol** (Sigma Aldrich, 459844-4L)
- **Qubit dsDNA HS kit** (Invitrogen, Q32854)
- **Qubit tubes** (Invitrogen, Q32856)
- **Nextera 96 plate** (Illumina)
- Various falcon tubes and tips
- **BrightLine™ Hemacytometer** (Sigma Aldrich)
- **LoBind clear 1.5 mL PCR clean** (Eppendorf, 03-395-565; 22343102)
#### Equipment
- **FACS Aria II Sorter** with 96 well plate holder
- **Ice buckets**
- **Refrigerated centrifuge** with 15 mL tube holders
- **ScreenTape** (Agilent)
- **Qubit** (Thermo)
#### N7-loaded Tn5
The original use of the protocol involved custom Tn5 loaded with Nextera N7 adapters (commercial equivalent example: Illumina FC-121-1030). Alternatively, unloaded Tn5 can be purchased and adapters loaded per the method described [here](https://www.biorxiv.org/content/10.1101/2019.12.17.879304v1.full.pdf).
## Protocol
### Buffer Preparation
1. **Nuclei Buffer:**
- Combine:
- 10 mM Tris-HCl (pH 7.4)
- 10 mM NaCl
- 3 mM MgCl2
- Store at 4°C.
2. **Nuclei Suspension Buffer (NSB):**
- 1 mL Nuclei Buffer
- 10 μL BSA
- 10 μL SUPERaseIn
- Chill on ice.
3. **Nuclei Buffer with BSA (NBB):**
- 1 mL Nuclei Buffer
- 10 μL BSA
- Chill on ice.
4. **10% Triton X-100 Stock:**
- 1 mL Triton X-100
- 9 mL Nuclease-free water.
- Store at 4°C.
5. **Permeabilization Buffer:**
- 500 μL per sample:
- 12.5 μL of 10% Triton X-100
- 487.5 μL NSB
- Pre-chill on ice.
### Permeabilization
6. **Thaw**
- Thaw frozen aliquots at 37°C in water bath.
7. **Buffer Addition**
- Add 400 μL of Permeabilization Buffer. Mix gently.
8. **Incubate**
- Incubate for 3 minutes on ice.
9. **Pellet and Resuspend**
- Pellet at 500g for 5 min (4°C), discard supernatant and resuspend.
10. **Recentrifuge**
- Pellet at 500g for 5 min (4°C), discard supernatant.
11. **Resuspend**
- Resuspend in 300 μL NSB and count nuclei with hemocytometer.
### Reverse Transcription
12. **Setup RT reaction**
- 30,000 nuclei in 22 μL Nuclei buffer
- 2 μL 10mM dNTP
- 2 μL indexed oligo-dT primer (100uM)
- Incubate at 55°C for 5 min, then cool on ice.
13. **Prepare RT Mix**
- 8 μL SuperScript IV First-Strand Buffer
- 2 μL 100mM DTT
- 2 μL SuperScript IV reverse transcriptase
- 2 μL RNaseOUT RNase Inhibitor
14. **Distribute RT mix and Incubate**
- Distribute 14 μL to each well. Incubate at following steps:
- 4°C for 2 mins
- 10°C for 2 mins
- 20°C for 2 mins
- 30°C for 2 mins
- 40°C for 2 mins
- 50°C for 2 mins
- 53°C for 15 mins
- 55°C for 10 mins
- Add 60 μL ice-cold NBB post reaction.
15. **Pool Nuclei**
- Pool Nuclei, pellet at 600 RCF for 10 min (4°C).
### Ligation
16. **Resuspend Nuclei**
- Resuspend nuclei in 1 mL NSB.
17. **Distribute and Add Indexing Oligos**
- Distribute 10 μL to each well, add 8 μL indexing oligos (100uM).
18. **Prepare Ligation Mix**
- Combine:
- 2 μL Quick Ligase
- 20 μL Quick Ligase buffer
- Distribute 22 μL to each well.
19. **Mix and Ligate**
- Mix by pipetting, then incubate at 25°C for 10 min.
- Add 60 μL NBB, pool all wells.
20. **Spin and Resuspend**
- Add 10 mL NBB, spin at 600 RCF, 10 min (4°C), supernatant discarded.
- Resuspend in 1 mL Elution Buffer.
21. **Add DAPI and Filter**
- Add 10 μL of 300 μM DAPI, mix gently.
- Filter through a 35 μM FACS tube.
### FACS Sorting
22. **Sort Nuclei**
- Add 4 μL Elution Buffer to each well, sort based on DAPI.
### Second Strand Synthesis
23. **Volume Check**
- Ensure volume in well is ~12 μL. Adjust input volumes if necessary.
24. **Prepare and Add Second Strand Mix**
- For each well, prepare:
- 1.33 μL second strand buffer
- 0.67 μL enzyme mix
- Add 2 μL per well.
25. **Incubate**
- Incubate at 16°C for 3 hours.
### Tagmentation
26. **Make TD Buffer**
- 1.2 mL tagmentation salt buffer
- 300 μL dimethylformamide
27. **Prepare Mix**
- 12.5 μL 2x TD buffer,
- 12.5 μL second tagmentation mix
- (optional: 0.02 μL N7 loaded Tn5)
- Incubate 5 min at 55°C.
### Ampure Bead Purification
28. **Add 50 μL of Ampure Beads**
- Incubate 5 min, transfer to magnet, incubate 3 min more.
29. **Wash beads**
- Twice with ~150 μL 80% ethanol.
30. **Resuspend Beads**
- Add 17 μL EB.
### Post-Bead Cleanup
31. **Bead Cleanup**
- With .7 volumes bead volume, wash with 80% ethanol.
### PCR
32. **Setup PCR**
- 2 μL indexed P5 PCR primer (10uM)
- 2 μL P7 primer (10uM)
- 20 μL NEBNext master mix
- PCR setting:
- 72°C 5 min
- 98°C 30 secs
- 17 cycles: 98°C 10 secs, 66°C 30 secs, 72°C 30 secs
- 72°C 5 min
### Quantify and Sequence
33. **Quantify & Sequence Sample**
- Quantified using Qubit and Agilent ScreenTape.
- Sequenced on Illumina Nextseq 2000, 100 bp kit.
- Read settings:
- Read 1: 34 bases
- Read 2: 66 bases
- Index 1: 10 bases
- Index 2: 10 bases
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