| ```markdown | |
| # 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 | |
| endofoutput | |
| ``` |