TITLE: UDA-5&#39;RNA-protocol
AUTHORS: Yun Li

[DESCRIPTION]
Droplet microfluidics-based single-cell combinatorial indexing sequencing represents an attractive way to balance cost, scalability, robustness, and accessibility. However, current methods need a tailored protocol for specific modality respectively, which may limit their potential for automation. We introduce UDA-seq, universal droplet microfluidics-based combinatorial indexing for massive-scale single-cell multimodal sequencing. We demonstrate that when necessary, UDA-seq enables effectively generating more than 100,000 single-cell data in a single-channel experiment of droplet microfluidics. Meanwhile, UDA-seq provides a universal workflow for accomplishing several multimodal tasks, including single-cell co-assay of RNA and VDJ, RNA and ATAC, and RNA and CRISPR guide RNA.

[STEPS]
SECTION: GEM Generation &amp; Barcoding.
1.     Prepare Master Mix & Load Chromium Next GEM Chip K
SECTION: GEM Generation &amp; Barcoding.
1.1. A certain number of cells or nuclei were added to Master Mix (18.8ul RT Reagent B (2000165); 7.3ul 1.1ul Template Switch Oligo (3000228); 1.9ul Reducing Agent B (2000087) and 8.3ul RT Enzyme C(2000085/2000102) each sample).
SECTION: GEM Generation &amp; Barcoding.
1.2. The microfluidic Chromium Next GEM Chip K (2000182) was loaded with 70 µl of cells or nuclei in thermoligation mix (inlet 1), 50 µl of Single Cell VDJ 5' Gel Bead (inlet 2, 10x Genomics catalog no. 1000264/1000267) and 45 µl of Partitioning Oil (inlet 3, 10x Genomics catalog no. 2000190) and run on the Chromium system.
SECTION: GEM Generation &amp; Barcoding.
1.3. The PCR mix was incubate in a thermomixer to perform enrichment PCR as follows: 53˚C for 45min, 4˚C hold.
SECTION: Pre-Amplification PCR
3.1. Prepare linear amplification mix(2X KAPA HiFi HotStart Ready Mix; 0.5uM Truseq-i5 index primer; 0.5uM Partial TSO/IS and 0.5uM P5 primer).
SECTION: Pre-Amplification PCR
3.2. Pipette 10X to mix thoroughly. Centrifuge briefly.
SECTION: Pre-Amplification PCR
3.3. PCR mix was incubate in a thermomixer to perform enrichment PCR as follows: 98˚C for 45s , and then 14 cycles of [ 98˚C for 20s, 63 °C for 30s, 72˚C for 1min]; 72˚C for 1min.
SECTION: Pre-Amplification PCR
3.4. cDNA PCR product were purified with 0.6x XP beads and elute in 300 ul EB.
SECTION: Pre-Amplification PCR
3.5. Purified with 0.7x XP beads and elute in 55 ul EB.
SECTION: 5ʹ Gene Expression (GEX) Library Construction
4.1. 50 ng mass of cDNA products (35ul) were mixed with 15 µl of i7-only TN5 Tagmentation Mix(10ul 5x Reaction Buffer (vazyme L buffer) and 5ul Self-i7 TN5).
SECTION: 5ʹ Gene Expression (GEX) Library Construction
4.2. Pipette mix 15x (pipette set to 30 µl) on ice. Centrifuge briefly.
SECTION: 5ʹ Gene Expression (GEX) Library Construction
4.3. Incubate in a thermal cycler with the following protocol. 55 ˚C for 10min.
SECTION: 5ʹ Gene Expression (GEX) Library Construction
4.4. PCR product were purified with 0.8x XP beads and elute in 40.5 ul EB.
SECTION: 5ʹ Gene Expression (GEX) Library Construction
5. GEX Sample Index PCR
SECTION: 5ʹ Gene Expression (GEX) Library Construction
5.1. Prepare and add Sample Index PCR Mix(NEBNex High-Fidelity 2X PCR Master Mix (NEB#M0541S); 0.5uM Partial P5 and 0.5uM Nextera P7-index). 
SECTION: 5ʹ Gene Expression (GEX) Library Construction
5.3. Incubate in a thermal cycler with the following protocol. 72 ˚C for 5 min, 98 ˚C for 45 s, 8-9 cycles of [98 ˚C for 20 s, 60 ˚C for 30 s, 72 ˚C for 1min], 72 ˚C for 5 min in thermocycler, storage at 4 ˚C.
SECTION: 5ʹ Gene Expression (GEX) Library Construction
5.4. PCR product were selected size with 0.6-0.8x XP beads and elute in 25.5 ul EB.
SECTION: VDJ Capture
6. VDJ capture 1
SECTION: VDJ Capture
6.1. Place a tube strip on ice and transfer 5 µl cDNA product.
SECTION: VDJ Capture
6.2. Prepare V(D)J Amplification 1 Reaction Mix(Amp Mix (2000047/2000103); 0.5uM Partial P5 and 0.5uM T/B VDJ outer primer in nuclease-free water) on ice. Vortex and centrifuge briefly.
SECTION: VDJ Capture
6.3. Add 75 µl V(D)J Amplification 1 Reaction Mix to each tube containing 5 µl sample.
SECTION: VDJ Capture
6.4. Pipette mix 5x (pipette set to 90 µl). Centrifuge briefly.
SECTION: VDJ Capture
6.5. Incubate in a thermal cycler with the following protocol. 98 ˚C for 45 s, 10 cycles for T cells /8 cycles for B cells of [98 ˚C for 20 s, 62 ˚C for 30 s, 72 ˚C for 1min], 72 ˚C for 1 min in thermocycler, storage at 4 ˚C.
SECTION: VDJ Capture
6.6. Store at 4°C for up to 72 h or proceed to the next step.
SECTION: VDJ Capture
6.7. PCR product were selected size with 0.5X-0.8x XP beads and elute in 30.5 ul EB.
SECTION: VDJ Capture
7. VDJ capture 2
SECTION: VDJ Capture
7.1. Prepare and add 50ul V(D)J Amplification 2 Reaction Mix(Amp Mix (2000047/2000103); 0.5uM Partial P5 and 0.5uM T/B VDJ inner primer) . 
SECTION: VDJ Capture
7.2. Pipette mix 5x (pipette set to 90 µl). Centrifuge briefly.
SECTION: VDJ Capture
7.3. Incubate in a thermal cycler with the following protocol. 98 ˚C for 45 s, 8 cycles for T cells /8 cycles for B cells of [98 ˚C for 20 s, 62 ˚C for 30 s, 72 ˚C for 1min], 72 ˚C for 1 min in thermocycler, storage at 4 ˚C.
SECTION: VDJ Capture
7.4. Store at 4°C for up to 72 h or proceed to the next step.
SECTION: VDJ Capture
7.5. PCR product were selected size with 0.5X-0.8x XP beads and elute in 30.5 ul EB.
SECTION: VDJ (GEX) LibraryConstruction
8.1. 50 ng mass of VDJ capture products (35ul) were mixed with 15 µl of i7-only TN5 Tagmentation Mix(10ul 5x Reaction Buffer (vazyme L buffer) and 5ul Self-i7 TN5).
SECTION: VDJ (GEX) LibraryConstruction
8.2. Pipette mix 15x (pipette set to 30 µl) on ice. Centrifuge briefly.
SECTION: VDJ (GEX) LibraryConstruction
8.3. Incubate in a thermal cycler with the following protocol. 55 ˚C for 5min.
SECTION: VDJ (GEX) LibraryConstruction
8.4. PCR product were purified with 0.8x XP beads and elute in 40.5 ul EB.
SECTION: VDJ (GEX) LibraryConstruction
9. GEX Sample Index PCR
SECTION: VDJ (GEX) LibraryConstruction
9.1. Prepare and add Sample Index PCR Mix(NEBNex High-Fidelity 2X PCR Master Mix (NEB#M0541S); 0.5uM Partial P5 and 0.5uM Nextera P7-index). 
SECTION: VDJ (GEX) LibraryConstruction
9.3. Incubate in a thermal cycler with the following protocol. 72 ˚C for 5 min, 98 ˚C for 45 s, 8 cycles of [98 ˚C for 20 s, 60 ˚C for 30 s, 72 ˚C for 1min], 72 ˚C for 5 min in thermocycler, storage at 4 ˚C.
SECTION: VDJ (GEX) LibraryConstruction
9.4. PCR product were selected size with 0.8x XP beads and elute in 25.5 ul EB.
SECTION: GEM clean-up
2.20. Transfer 17 µl sample to a Pre-Amplification PCR.
SECTION: GEM clean-up
2.19. Place on the magnet•Low until the solution clears.
SECTION: GEM clean-up
2.18. Incubate 1 min at room temperature.
SECTION: GEM clean-up
2.17. Vortex for 15 sec to mix thoroughly. 
If beads still appear clumpy, continue pipette mixing until fully resuspended.
SECTION: GEM clean-up
2.16. Remove from the magnet. Immediately add 17.5 µl Elution Solution I.
SECTION: GEM clean-up
2.15. Centrifuge briefly. Remove remaining ethanol. Air dry for 2 min.
SECTION: GEM clean-up
2.14. Remove the ethanol.
SECTION: GEM clean-up
2.13. Add 200 µl 80% ethanol to pellet. Wait 30 sec.
SECTION: GEM clean-up
2.12. Remove the ethanol.
SECTION: GEM clean-up
2.11. Add 300 µl 80% ethanol to the pellet while on the magnet. Wait 30 sec.
SECTION: GEM clean-up
2.10. Remove the supernatant.
SECTION: GEM clean-up
2.9. At the end of 10 min incubation, place on a on a 96-well plate magnet until the solution clears.
SECTION: GEM clean-up
2.8. Prepare Elution Solution I(1960ul Buffer EB; 20ul 10% Tween 20 and 20ul Reducing Agent B(2000087)). Vortex and centrifuge briefly.
SECTION: GEM clean-up
2.7. Incubate 10 min at room temperature.
SECTION: GEM clean-up
2.6. Pure the samples with 16ul Dynabeads Cleanup Mix(40ul Nuclease-free Water; 1465ul Cleanup Buffer(2000088); 64ul Dynabeads MyOne SILANE(2000048) and 40ul Reducing Agent B(2000087), vortex for 15 sec to mix thoroughly.
SECTION: GEM clean-up
2.5. Add 6ul EB.
SECTION: GEM clean-up
2.4. Incubate 85˚C for 5min.
SECTION: GEM clean-up
2.3. Add 150ul PBS to the remaining aqueous phase, mix well then dispense the liquid evenly into 96-well plate, each well add 2ul.
After brief centrifugation, the products can be stored at −80 °C for at least 2 weeks.
SECTION: GEM clean-up
2.2. Slowly remove and discard 125 µl Recovery Agent/Partitioning Oil (pink) from the bottom of the tube. DO NOT aspirate any aqueous sample.
SECTION: GEM clean-up
2.1. Add 125 µl Recovery Agent to each sample (post GEM-RT incubation) at room temperature. 
DO NOT pipette mix or vortex the biphasic mixture. Wait 2 min.
SECTION: GEM clean-up
2. Cleanup – Dynabeads
SECTION: Pre-Amplification PCR
3. cDNA index amplification
SECTION: 5ʹ Gene Expression (GEX) Library Construction
4. Fragmentation
SECTION: 5ʹ Gene Expression (GEX) Library Construction
5.2. Pipette mix and centrifuge briefly.
SECTION: VDJ (GEX) LibraryConstruction
8. Fragmentation
SECTION: VDJ (GEX) LibraryConstruction
9.2. Pipette mix and centrifuge briefly.