TITLE: LungMAP2 URMC cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) protocol
AUTHORS: Jeffrey Malik, Gloria S Pryhuber, John Ashton, Ravi Misra, Cameron Baker, Gautam Bandyopadhyay, Heidie Huyck

[DESCRIPTION]
The LungMAP program has been working for over a decade to provide the lung community with a comprehensive resource for the lung research community, including reference data sets and protocols [1]. The University of Rochester has served as the Human Tissue Core and worked extensively with Research Center lab groups. We have established protocols for procuring human tissue including, but not limited to lung, and processing them for a variety of downstream analysis modalities including the generation of single cell suspensions [2, 3, 4]. We have successfully utilized singe cells suspensions to generate single cell CITESeq data utilizing the 10X Genomics Single Cell 3' (v3.1) RNA sequencing platform [5-7]. This protocol describes the steps necessary for preparing generating sequencing data following staining single cells with ADT-tagged antibodies (including library preparation, sequencing steps, and mapping steps).





References 
1. https://www.lungmap.net/about-lungmap/lungmap-objectives/

2. 602.2 Donor Acceptance Criteria for URMC HTC SenNet Inclusion dx.doi.org/10.17504/protocols.io.biz7kf9n.

3. 603.3 & 604.5_URMC_HTC_Lung and Lobe Processing for SenNet dx.doi.org/10.17504/protocols.io.n2bvj395plk5/v1

4. 702.B.3 URMC HTC Lung Tissue Digestion SOP + Worksheet 052820 dx.doi.org/10.17504/protocols.io.biz5kf86

5. 709.1 Staining of Dissociated Lung Cells for Cellular Indexing of Transcriptomes and Epitopes by Sequencing (CITE sequencing) V.2 dx.doi.org/10.17504/protocols.io.e6nvw1jk7lmk/v2

6. TotalSeq‱-A Antibodies and Cell Hashing with 10x Single Cell
3' Reagent Kit v3 3.1 Protocol dx.doi.org/10.17504/protocols.io.8aahsae

7. Chromium Next GEM Single Cell 3’ Reagent Kits v3.1(Dual Index) User Guide. Document Number: CG000315. October 2022.
 

[GUIDELINES]
Full protocol is from 10X Genomics. All modifications are to the original protocol (Chromium Next GEM Single Cell 3' Reagent Kits v3.1 User Guide (Rev E) CG000315, support.10xgenomics.com) and TotalSeq‱-A Antibodies and Cell Hashing with 10x Single Cell 3' Reagent Kit v3.1 (Dual Index) Protocol (https://www.biolegend.com/en-us/protocols/totalseq-a-dual-index-protocol).

[STEPS]
SECTION: GEM Generation and Barcoding
1. Prepare Reagents for use 
Equilibrate gel beads to room temperature for 30 min. 
Thaw, vortex, and centrifuge RT Reagent, Template Switch Oligo, and Reducing Agent B. Verify no precipitate in Reducing Agent B. 
Centrifuge RT Enzyme C before adding to Master Mix.

SECTION: GEM Generation and Barcoding
2. Prepare Master Mix
Prepare on ice. Pipette mix 15x and centrifuge briefly. 
Add 31.9 µL Master Mix to each tube of a PCR 8-tube strip on ice.

SECTION: GEM Generation and Barcoding
3. Load Chromium Single Cell G Chip 
1.  Assemble Next GEM Chromium Chip G in a 10X Chip Holder.
2.  Must load chip in order according to row label for microfluidic channels to work properly: 
     Row 1 → Row  2 → Row 3 
3.  Make sure NO bubbles are introduced while loading chip. 
4.  Dispense 50% glycerol solution into unused Chip Wells (if <8 samples used per chip) 
          a.	75 µL into unused wells in Row 1 
          b.  40 µL into unused wells in Row 2 
          c.	45 µL into unused wells in Row 3 
DO NOT add 50% glycerol solution to the bottom row of NO FILL wells. DO NOT use any substitute for 50% glycerol solution.
5.  Use Cell Suspension Volume Calculator Table (see step 1.2 of Chromium Next GEM Single Cell 3' v3.1 protocol) to add the appropriate volume of nuclease-free water to Master Mix already in PCR 8-tube strip.  Add corresponding volume of gently pipette-mixed single nuclei suspension to Master Mix.  Total of 75 µL in each tube.  Gently pipette mix, avoid introducing bubbles.   DO NOT add nuclease free-water directly to single cell suspension, add instead to Master Mix. 
6.	Cell Loading 
          a.load 32,000 nuclei → target recovery 20,000
7. Load70 µL Master Mix + Nuclei Suspension into the bottom center of each well in Row 1 without introducing bubbles. 
8.	Snap the Gel Bead strip into a 10X Vortex Adapter. Vortex 30 s. Remove the Gel Bead strip and flick sharply downward to ensure maximum recovery. Confirm there are no bubbles at the bottom of the tubes and liquid levels look even. 
10.	Load 50 µL gel beads into wells of Row 2. Only puncture the foil seal for gel bead tubes being used. Dispense slowly and without introducing bubbles. 
11.	Load45 µL partitioning oil into each Row 3 by pipetting two aliquots of 140 µL. 
12.	Attach 10X gasket. Align the top-notch. Ensure gasket holes are aligned with the wells. Avoid touching the smooth gasket surface. Do not press down on the gasket. Run chip immediately after loading the partitioning oil.
SECTION: GEM Generation and Barcoding
4. Run the Chromium Controller
Press the eject button on the controller to eject tray.
Place the assembled chip with the gasket in the tray. Press the button to retract the tray.
Confirm the program on screen. Press the play button. 
At completion of the run (~18 min), the controller will chime. Proceed immediately to the next step.
Firmware Version 4.0 or higher is required in the Chromium Controller or the Chromium Single Cell Controller used for this protocol.

SECTION: GEM Generation and Barcoding
5. Transfer GEMs 
Chill strip tubes on ice. 
Press the eject button to remove the chip. 
Discard the gasket. Open the chip holder. Fold the lid back until it clicks to expose the wells at 45 degrees. 
Check the volume in rows 1-2. Abnormally high volume in any well indicates a clog. 
Slowly aspirate 100 uL GEMs from the lowest points of the Recovery Wells without creating a seal between the pipette tips and the wells. 
 

6.  Slowly dispense (~20 s) GEMs into the tube strip on ice with the pipette tips against the sidewalls of the wells.
If multiple chips are run back-to-back, cap/cover the GEM-containing tube strip and place on ice for no more than 1 h.

SECTION: GEM Generation and Barcoding
6. GEM-RT Incubation
Set up the cDNA reaction.
Incubate in a thermocycler to complete reverse transcription (53 °C 45 min , 85 °C 5 min , 4 °C HOLD). 
Store at 4 °C for up to 4320 minor at -20 °C for up to a week. Or proceed to the next step.

SECTION: GEM Generation and Barcoding
7. Post GEM-RT Cleanup & cDNA Amplification - Prepare Reagents for use (See 10X User Guide for details) 
Thaw, vortex, and centrifuge a tube of Reducing Agent B and cDNA primers.
Maintain Amp Mix on ice after vortex and centrifuge 
Thaw Cleanup Buffer at 65 °C for 10 min with shaking at max rpm then cool to room temperature. Verify no visible crystals. Cool to RT.

SECTION: GEM Generation and Barcoding
8. Post GEM RT-Cleanup --Dynabead 
Add125 µL Recovery Agent to each sample at room temperature. DO NOT MIX. Wait 2 min. 
Carefully aspirate 125 µL of pink oil phase from the bottom of the tube. DO NOT aspirate any aqueous sample. 
Prepare Dynabeads Cleanup Mix (See 10X User Guide for detail)
Vortex and briefly centrifuge mixture. Add 200 µL to each sample. Pipette 10x to mix.
Incubate at room temperature for 10 min; pipette mix again 5 min into incubation. 
Prepare Elution Solution I (See User Guide for Detail). Vortex and centrifuge briefly. 
Place sample on magnetic separator-HIGH until solution clears. Remove the supernatant (acqueous phase and Recovery Agent). Add 300 µL  freshly prepared 80% ethanol. Wait 30 sec. Remove Ethanol. Repeat.Remove the ethanol.
Centrifuge briefly. Place on the magnet•Low. Remove remaining ethanol. Air dry for 1 min.
Remove from the magnet. Immediately add 35.5 µl Elution Solution I (prepared in
step above). Pipette mix (pipette set to 30 µl) without introducing bubbles.
10. Incubate 2 min at room temperature.
11. Place on the magnet•Low until the solution clears.   Transfer 35 µl sample to a new tube strip.

SECTION: GEM Generation and Barcoding
9. cDNA Amplification with the addition of ADT and HTO primers
Prepare cDNA Amplification Mix on ice (see 10x User Guide for details and Step 16 of TotalSeq‱-A Antibodies and Cell Hashing with 10x Single Cell 3' Reagent Kit v3.1 (Dual Index) Protocol dx.doi.org/10.17504/protocols.io.8aahsae). Vortex and centrifuge briefly. 
Add 65 µL cDNA Amplification Reaction Mix including ADT and HTO primers to 35 µL sample.
Pipette mix 15x to mix and centrifuge briefly.
Incubate in a thermocycler to amplify cDNA.
 



SECTION: GEM Generation and Barcoding
10. cDNA Cleanup --SPRIselect 
Perform a 0.6X beads purification with SPRIselect reagent (60 µL )
Incubate x 5 minutes at RT.
Place on magent-HIGH until solution clears. Transfer 70 ul of supernatant to a new tube. Note: the ADT/HTO material will be in the supernatant and the cellular RNA expression pool will be on the SPRI beads. Library construction will be completed in parallel  with 3' Gene Expression Library Construction 
Add 200 µL freshly prepared 80% ethanol, wait 30 sec and remove ethanol.  Repeat.
Place on magent-HIGH until solution clears. Transfer  EB. Pipette mix x 15x. Incubate 2 min RT.
Place on magent-HIGH until solution clears. Transfer40 µL to a new strip tube.
Store at 4 °C for up to 4320 min or at -20 °C for up to 4 weeks. Or proceed to the next step.



SECTION: GEM Generation and Barcoding
11. cDNA QC & Quantification
Run TapeStation to obtain concentration and size 
Expected size range for amplified cDNA: 400 to 2500 bp 
cDNA total yield range: 80 to 1000 ng
 

SECTION: 3&#x27; Gene Expression Library Construction
12. Prepare Reagents for use 
Thaw, vortex, and centrifuge Fragmentation Buffer, Adaptor Oligos, Ligation Buffer, SI Primer. Verify no precipitate.
Maintain on ice Fragmentation Enzyme, DNA Ligase, and Amp Mix.
SECTION: 3&#x27; Gene Expression Library Construction
13. Fragmentation, End Repair & A-tailing
1.  Prepare Fragmentation Mix on ice (See User Manual for volume details). Pipette mix and centrifuge briefly. 
2.  Transfer 10 µL purified cDNA to a new strip tube 

 

3.  Add 25 µL Buffer EB to each sample
4.  Add 15 µL Fragmentation Mix to each sample
5.  Pipette mix, centrifuge briefly, and transfer to pre-cooled block. “SKIP” hold step to initiate the protocol

SECTION: 3&#x27; Gene Expression Library Construction
14. Post Fragmentation, End Repair & A-tailing Double Sided Size Selection --SPRIselect 
Vortex to resuspend the SPRIselect reagent. Add 30 µL SPRIselect reagent (0.6X) to each sample and pipette mix 15x. 
Incubate 5 min at room temperature.
Centrifuge briefly. 
Place sample on magnetic separator-HIGH until solution clears. DO NOT discard supernatant.  
Transfer 75 µL supernatant to a new tube strip tube. 
Vortex to resuspend the SPRIselect reagent. Add 10 µL SPRIselect reagent (0.8X) to each transferred supernatent and pipette mix 15x. 
Incubate 5 min RT
Place sample on magnetic separator-HIGH until solution clears. 
Remove 80 µL supernatant. DO NOT discard any beads. 
Wash twice with 125 µL freshly prepared 80% ethanol. 
Centrifuge briefly. Place on magent-LOW until solution clears. Remove remaining ethanol, do not overdry.
Remove from magnet. Add 50.5 µL Qiagen Buffer EB, pipette mix x 15. Incubate x 2 min RT.
Place on magent-HIGH until clear. Transfer 50 µL to a new strip tube.
SECTION: 3&#x27; Gene Expression Library Construction
15. Adaptor Ligation
Prepare Adaptor Ligation Mix on ice. Pipette mix and centrifuge briefly.
Add 50 µL Adaptor Ligation Mix to sample. Pipette mix 15x. Centrifuge briefly.
Incubate in a thermocycler to ligate adaptor. (20 °C x 15 min , 4 °C Hold)

SECTION: 3&#x27; Gene Expression Library Construction
16. Post Ligation Cleanup --SPRIselect 
Perform a 0.8X beads purification with SPRIselect reagent (add 80 µL reagent to each sample, pipette-mix x 15x, incubate 5 min at RT)
Place on magnet-HIGH until solution clears, remove supernatant. Wash twice with freshly prepared 80% ethanol (add 200 µL, wait 30 sec, remove ethanol and repeat)
Centrifuge briefly. Place on magent-LOW. Remove remaining ethanol and air dry up to 2 min.
Remove from magnet. To elute, add 30.5 µL Qiagen Buffer EB. Pipette mix x 15. Incubate 2 min at RT.  Place on magent-LOW until solution clears.
Transfer 30 µL to a new strip tube.
SECTION: 3&#x27; Gene Expression Library Construction
17. Sample Index PCR
1.  Choose the appropriate sample index sets to ensure that no sample indices overlap in a multiplexed sequencing run. 
2.  Prepare Sample Index PCR Mix on ice. Pipette mix and centrifuge briefly.
3.  Add 50 µL Amp Mix to the 30 µL sample already in the new strip tube.
4.  Add 20 µL of an individual Dual Index TT Set A to each sample. Record well assignment. Pipette-mix x5 and centrifuge briefly. 
5.  Incubate in a thermocycler to incorporate sample indices. 
 

6.  Store at 4 °C for up to 4320 min. Or proceed to the next step.

SECTION: 3&#x27; Gene Expression Library Construction
18. Post Sample Index PCR Double Sided Size Selection --SPRIselect 
Vortex to resuspend the SPRIselect reagent. Add 60 µL SPRIselect reagent (0.6X) to each sample and pipette mix 15x. 
Incubate 5 min at room temperature. 
Centrifuge briefly 
Place sample on magnetic separator. DO NOT discard supernatant. 
Transfer 150 µL supernatant to a new tube strip tube. 
Vortex to resuspend the SPRIselect reagent. Add 20 µL SPRIselect reagent (0.8X) to each sample and pipette mix 15x. 
Incubate 5 min at room temperature.
Centrifuge briefly 
Place sample on magnetic separator-HIGH until solution clears. 
Remove 165 µL supernatant. DO NOT discard any beads. 
With tube still on magnet, add 200 µLfreshly prepared 80% ethanol to pellet. Wait 30 sec and then remove ethanol.
Repeat Step 11 for total of 2 washes then centrifuge briefly. Place on magnet-LOW. Remove remaining ethanol.	
Remove from magnet. Add 35.5 µL Qiagen Buffer EB. Pipette-mix x 15)
Incubate 2 min at RT.
Place on magnet-LOW until solution clears then transfer 35 µL to a new strip tube. 
Store at 4 °C for up to 4320 min or at -20 °C for long-term storage.
 

SECTION: Sequencing
37. NovaSeq Sequencing (target - 100,000 reads per nucleus)
1.  Paired End, Dual Indexing 
        a.  Read 1: 28 cycles 
        b.  i7 Index: 10 cycles
        c.  i5 Index: 10 cycles 
        d.  Read 2: 91 cycles
2.  Library Loading 
        a.  10X recommended Loading concentration: 300 pM 
        b.  Optional: 1% PhiX
3.  Output
        a.  SP: 650–800 million reads
        b.  S1: 1.3–1.6 billion reads 
        c.  S2: 3.3 –4.1 billion reads
         d.  S4: 8-10 billion reads
SECTION: Amplify Dual-Indexed ADT Sequencing Library
19. Aliquot 22.5 uL of each sample (ADT cDNA) to a new, labeled 0.5-mL strip tube.
SECTION: Amplify Dual-Indexed ADT Sequencing Library
20. Add 25 uL of 2X Q5 HotStart HiFi Master Mix to each sample.
SECTION: Amplify Dual-Indexed ADT Sequencing Library
21. Add 2.5 uL of appropriate indexed primer-pair (10uM each primer) to each sample. Mix by pipetting and spin briefly.
SECTION: Amplify Dual-Indexed ADT Sequencing Library
22.  Step  Temperature    Time    
1          95°C                Hold    
2          95°C                3:00     
3          95°C                0:20     
4          60°C                0:30     
5          72°C                0:20     
6          Complete 12 cycles                                    
7          72°C                5:00     
8          4°C                  Hold    

 
SECTION: Amplify Dual-Indexed ADT Sequencing Library
23. Add 60 ul AMPure beads (1.2X ratio) to each sample and pipette to mix 10X
SECTION: Amplify Dual-Indexed ADT Sequencing Library
24. Incubate 5 minutes at room temperature.
SECTION: Amplify Dual-Indexed ADT Sequencing Library
25. Place tube on magnet and wait 1 minute until solution is clear.
SECTION: Amplify Dual-Indexed ADT Sequencing Library
26. Carefully remove and discard the supernatant.
SECTION: Amplify Dual-Indexed ADT Sequencing Library
27. Add 200 μl 80% Ethanol to the tube without disturbing the pellet and stand for 30 seconds (first Ethanol wash).
SECTION: Amplify Dual-Indexed ADT Sequencing Library
28. Carefully remove and discard the ethanol wash.
SECTION: Amplify Dual-Indexed ADT Sequencing Library
29. Add 200 μl 80% Ethanol to the tube without disturbing the pellet and stand for 30 seconds (second Ethanol wash).
SECTION: Amplify Dual-Indexed ADT Sequencing Library
30. Carefully remove and discard the ethanol wash.
SECTION: Amplify Dual-Indexed ADT Sequencing Library
31. Centrifuge tube briefly and return it to magnet.
SECTION: Amplify Dual-Indexed ADT Sequencing Library
32. Remove and discard any remaining ethanol and allow the beads to air dry for 2 minutes.
SECTION: Amplify Dual-Indexed ADT Sequencing Library
33. Resuspend beads in 32 μl EB.
SECTION: Amplify Dual-Indexed ADT Sequencing Library
34. Pipette mix vigorously and incubate at room temperature for 5 minutes.
SECTION: Amplify Dual-Indexed ADT Sequencing Library
35. Place tube on magnet and transfer 30 uL supernatant to low-bind tube.
SECTION: Amplify Dual-Indexed ADT Sequencing Library
36. Quantify library by standard methods (Qubit & BioAnalyzer). ADT library will be around 210 bp.
SECTION: Cell Ranger Mapping and Analysis Pipeline
38. Create a feature reference file corresponding to 
the antibody panel and library mapping file corresponding to the file 
paths for the ADT and GEX libraries. Run cellranger count, incorporating
 both of these files and the combined Human+COVID reference
SECTION: Cell Ranger Mapping and Analysis Pipeline
39. Create fastq from bcl files using cellranger mkfastq with default parameters
SECTION: Cell Ranger Mapping and Analysis Pipeline
40. Insert 10X provided well indices into Clarity LIMS generated sample sheet.
SECTION: Cell Ranger Mapping and Analysis Pipeline
41. Create combined human and COVID reference by downloading the Human 2020-A reference from https://www.10xgenomics.com/support/software/cell-ranger/latest/release-notes/cr-reference-release-notes#2020-a. Download the full COVID sequence from https://www.ncbi.nlm.nih.gov/nuccore/MT547814. Append the COVID fasta onto the human genome fasta. Create a record within the human GTF file corresponding to the full COVID sequence.