TITLE: Single Cell RNA sequencing (scRNAseq) of fresh human lung cell suspension
AUTHORS: Julian Alonso Chamucero, Mauricio Rojas, Ana Mora

[DESCRIPTION]
Gene expression analysis is a molecular biology approach that allows, among other things, to evaluate the differential expression of genes involved in the development of a certain disease and to identify possible therapeutic targets. The lung is a complex organ composed of more than 52 different cell types, which makes it necessary to implement cutting-edge technologies such as single cell RNA seq analysis for its study. This approach allows to identify genes potentially associated with the development and/or progression of age-related diseases as the idiopathic pulmonary fibrosis, and to identify genes associated with the senescence process and to correlate their expression in certain cell types and their involvement in those diseases. This protocol allows the dissociation of a human lung specimen to a cell suspension and the generation of single cell 3´gene expression dual index libraries from single cells. 

[BEFORE_START]
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[GUIDELINES]
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[STEPS]
SECTION: Tissue Dissociation
2. Soak the lung pieces in PBS (3x) to remove red blood cells.
SECTION: Tissue Dissociation
3. Compress with a sterile gauze pad to remove excess liquid.
SECTION: Tissue Dissociation
4. Carefully remove the parietal pleura.
SECTION: Tissue Dissociation
5. Dissect the tissue into 1-cm3 pieces and transfer to a 50 ml conical tube (15 g of tissue without pleura per tube) containing the enzyme cocktail (1 mg/ml liberase DL, DNase I, DMEM).
SECTION: Tissue Dissociation
6. Allow the sample to digest for 2 hours at 37°C.
SECTION: Tissue Dissociation
7. Inactive the digestion buffer (collagenolytic and proteolytic activity) with 2 ml of cold FBS and leave the sample on ice for 5 minutes. 
SECTION: Tissue Dissociation
8. Serially filter the suspension through 300-μm, 100-μm, 70-μm and strainers. 
SECTION: Tissue Dissociation
9. After straining, centrifuge 500g for 7 minutes.
SECTION: Tissue Dissociation
10. Remove supernatant, add 1x RBC lysis to pellet, re-suspend pellet.
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11. Incubate in 4 degree C fridge for 7 minutes.
SECTION: Tissue Dissociation
12. Add PBS (10% FBS), centrifuge 500g for 7 minutes.
SECTION: Tissue Dissociation
13. Remove supernatant, re-suspend pellet in 10ml PBS.
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14. Filter through 40-μm strainer one more time to remove clumped, dead cells.
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15. Count cells and viability using countess automatic cell counter, confirm reading under themicroscope.
SECTION: Tissue Dissociation
16. Prepare a vial with a cell stock concentration of 1000 cells/ul and keep on ice.
SECTION: Tissue Dissociation
17. Proceed to the GEM generation and barcoding step
SECTION: GEM generation and barcoding
18. Prepare Master Mix on ice: add reagents in the order listed. Pipette mix 15x and centrifuge briefly.

 
 
     Reagent      1X (ul)      2X (ul)      3X (ul)      4X (ul)      5X (ul)      6X (ul)      7X (ul)      8X (ul)        RT Reagent B      18.8      41.4      62.0      82.7      103.4      124.1      144.8      165.4        Template Switch Oligo      2.4      5.3      7.9      10.6      13.2      15.8      18.5      21.1        Reducing Agent B      2.0      4.4      6.6      8.8      11.0      13.2      15.4      17.6        RT Enzyme C      8.7      19.1      28.7      38.3      47.9      57.4      67.0      76.6        Total      31.9      70.2      105.3      140.4      175.5      210.5      245.6      280.7   

 
 
SECTION: GEM generation and barcoding
19. Add 31.9 µl Master Mix into each tube of a PCR 8-tube strip on ice.
SECTION: GEM generation and barcoding
20. Assemble Chromium Next GEM Chip G:
SECTION: GEM generation and barcoding
20.1. After removing the chip from the sealed bag, use the chip in ≤ 24 h.
SECTION: GEM generation and barcoding
20.2. Align notch on the chip (upper left corner) and the holder.
SECTION: GEM generation and barcoding
20.3. Insert the left-hand side of the chip under the guide. Depress the righthand side of the chip until the spring-loaded clip engages.
SECTION: GEM generation and barcoding
20.4. Close the lid before dispensing reagents into the wells.
SECTION: GEM generation and barcoding
21. Load Chromium NextGEM Chip G:
SECTION: GEM generation and barcoding
21.1. a)Add 50% glycerol solution to each unused well (if processing <8 samples/chip).
    i)70 µl in each unused well in row labeled 1.
    ii)50 µl in each unused well in row labeled 2.
    iii)45 µl in each unused well in row labeled 3.
SECTION: GEM generation and barcoding
22. Prepare Master Mix + Cell suspension:
SECTION: GEM generation and barcoding
22.1. Refer to the Cell Suspension Volume Calculator Table
SECTION: GEM generation and barcoding
22.2. Add the appropriate volume of nuclease-free water to Master Mix. Pipette mix 5x. Add corresponding volume of single cell suspension to Master Mix. Total of 75 µl in each tube.
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22.3. Gently pipette mix the cell suspension before adding to the Master Mix.
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23. Load Row Labeled 1:
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23.1. Gently pipette mix the Master Mix + Cell Suspension.
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23.2. Using the same pipette tip, dispense 70 µl Master Mix + Cell Suspension into the bottom center of each well in row labeled 1 without introducing bubbles.
SECTION: GEM generation and barcoding
24. Prepare Gel Beads:
SECTION: GEM generation and barcoding
24.1. Snap the tube strip holder with the Gel Bead strip into a 10x Vortex Adapter. Vortex 30 sec.
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24.2. Centrifuge the Gel Bead strip for ~5 sec.
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24.3. Confirm there are no bubbles at the bottom of the tubes and the liquid levels are even.
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24.4. Place the Gel Bead strip back in the holder. Secure the holder lid.
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25. Load Row Labeled 2:
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25.1. Puncture the foil seal of the Gel Bead tubes.
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25.2. Slowly aspirate 50 µl Gel Beads.
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25.3. Dispense into the wells in row labeled 2 without introducing bubbles.
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26. Load Row Labeled 3:
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26.1. Dispense 45 µl Partitioning Oil into the wells in row labeled 3 from a reagent reservoir.
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27. Attach GEM Gasket
SECTION: Tissue Dissociation
1. Collect 15 to 30 g of lung tissue.
SECTION: GEM generation and barcoding
27.1. Align the notch with the top left-hand corner.
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27.2. Ensure the gasket holes are aligned with the wells.
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28. Run the Chromium Controller:
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28.1. Press the eject button on the Controller to eject the tray.
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28.2. Place the assembled chip with the gasket in the tray, ensuring that the chip stays horizontal. Press the button to retract the tray
SECTION: GEM generation and barcoding
28.3. Confirm the Chromium Chip G program on screen. Press the play button.
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28.4. At completion of the run (~18 min), the Controller will chime. Immediately proceed to the next step.
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29. Transfer GEMs:
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29.1. Place a tube strip on ice.
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29.2. Press the eject button of the Controller.
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29.3. Discard the gasket. Open the chip holder. Fold the lid back until it clicks to expose the wells at 45 degrees.
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29.4. Slowly aspirate 100 µl GEMs from the lowest points of the recovery wells in the top row labeled 3 without creating a seal between the tips and the bottom of the wells.
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29.5. Over the course of ~20 sec, dispense GEMs into the tube strip on ice with the pipette tips against the sidewalls of the tubes.
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30. GEM-RT incubation:
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30.1. a)Incubate in a thermal cycler with the following protocol:
 
     Lid Temperature      Reaction Volume      Run time        53°C      125 ul      ~55 min        Step      Temperature      Time        1      53°C      0:45:00        2      85°C      0:05:00        3      4°C      Hold   
 
SECTION: GEM generation and barcoding
30.2. Store at 4°C for up to 72 h or at −20°C for up to a week or proceed to the next step.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
31. Post GEM-RT Cleanup – Dynabeads:
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
31.1. a)Add 125 µl Recovery Agent to each sampleat room temperature. DO NOT pipette mix or vortex the biphasic mixture. Wait 2 min.
    i) The resulting biphasic mixture contains Recovery Agent/Partitioning Oil (pink) and aqueous phase (clear), with no persisting emulsion (opaque).
    ii) If biphasic separation is incomplete: Firmly secure the cap on the tube strip, ensuring that no liquid is trapped between the cap and the tube rim. Mix by inverting the capped tube strip 5x, centrifuge briefly, and proceed to step b.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
31.2. Slowly remove and discard 125 µl Recovery Agent/Partitioning Oil (pink) from the bottom of the tube. DO NOT aspirate any aqueous sample.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
31.3. a)Prepare Dynabeads Cleanup Mix:

     Reagent      1X (ul)      2X (ul)      3X (ul)      4X (ul)      5X (ul)      6X (ul)      7X (ul)      8X (ul)        Cleanup buffer      182      400      601      801      1001      1201      1401      1602        Dynabeads MyOne SILANE      8      18      26      35      44      53      62      70        Reducing Agent B      5      11      17      22      28      33      39      44        Nuclease-free Water      5      11      17      22      28      33      39      44        Total      200      440      660      880      1100      1320      1540      1760   

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31.4. Vortex and add 200 µl to each sample. Pipette mix 10x (pipette set to 200 µl).
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31.5. Incubate 10 min at room temperature (keep caps open). Pipette mix again at ~5 min after start of incubation to resuspend settled beads
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
31.6. a)Prepare Elution Solution I. Add reagents in the order listed. Vortex and centrifuge briefly.

     Reagent      2X (ul)      4X (ul)      6X (ul)      8X (ul)        Buffer EB      98      196      294      392        10% Tween 20      1      2      3      4        Reducing Agent B      1      2      3      4        Total      100      200      300      400   

SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
31.7. At the end of 10 min incubation, place on a 10x Magnetic Separator•High position (magnet•High) until the solution clears.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
31.8. Remove the supernatant (aqueous phase and Recovery Agent).
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
31.9. Add 300 µl 80% ethanol to the pellet while on the magnet. Wait 30 sec.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
31.10. Remove the ethanol.
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31.11. Add 200 µl 80% ethanol to pellet. Wait 30 sec.
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31.12. Remove the ethanol.
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31.13. Centrifuge briefly. Place on the magnet•Low.
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31.14. Remove remaining ethanol. Air dry for 1 min.
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31.15. Remove from the magnet. Immediately add 35.5 µl Elution Solution I.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
31.16. Pipette mix (pipette set to 30 µl) without introducing bubbles.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
31.17. Incubate 2 min at room temperature.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
31.18. Place on the magnet•Low until the solution clears
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31.19. Transfer 35 µl sample to a new tube strip.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
32. cDNA amplification:
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
32.1. a)Prepare cDNA Amplification Mix on ice. Add reagents in the order listed. Vortex and centrifuge briefly.

     Reagent      1X (ul)      2X (ul)      3X (ul)      4X (ul)      5X (ul)      6X (ul)      7X (ul)      8X (ul)        Amp Mix      50      110      165      220      275      330      385      440        cDNA Primers      15      33      50      66      83      99      116      132        Total      65      143      215      286      358      429      501      572   

SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
32.2. Add 65 µl cDNA Amplification Reaction Mix to 35 µl sample.
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32.3. Pipette mix 15x (pipette set to 90 µl). Centrifuge briefly.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
32.4. a)Incubate in a thermal cycler with the following protocol:

     Lid   Temperature      Reaction   Volume      Run time        105°C      100 ul      ~30-45   min        Step      Temperature      Time        1      98°C      0:03:00        2      98°C      0:00:15        3      63°C      0:00:20        4      72°C      0:01:00        5      11   Cycles *        6      72°C      0:01:00        7      4°C      Hold        *   Targeted cell recovery >6,000   

SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
32.5. Store at 4°C for up to 72 h or −20°C for ≤1 week or proceed to the next step.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
33. cDNA Cleanup – SPRIselect:
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
33.1. Vortex to resuspend the SPRIselect reagent. Add 60 µl SPRIselect reagent (0.6X) to each sample and pipette mix 15x (pipette set to 150 µl).
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
33.2. Incubate 5 min at room temperature.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
33.3. Place on the magnet•High until the solution clears.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
33.4. Remove the supernatant.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
33.5. Add 200 µl 80% ethanol to the pellet. Wait 30 sec.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
33.6. Remove the ethanol.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
33.7. Repeat steps 5 and 6 for a total of 2 washes.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
33.8. Centrifuge briefly and place on the magnet•Low.
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33.9. Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will decrease elution efficiency.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
33.10. Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will decrease elution efficiency.
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33.11. Incubate 2 min at room temperature.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
33.12. Place the tube strip on the magnet•High until the solution clears.
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33.13. Transfer 40 µl sample to a new tube strip.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
33.14. Store at 4°C for up to 72 h or at −20°C for up to 4 weeks, or proceed to the next step.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
34. cDNA Quantification:
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
34.1. Run 2 µl sample with the Qubit dsDNA HS Assay Kit.
SECTION: Post GEM–RT Cleanup &amp; cDNA Amplification
34.2. Multiply the cDNA concentration reported via the Qubit Fluorometer by the elution volume (40 µl) to obtain the total cDNA yield in ng.
SECTION: 3’ Gene Expression Dual Index Library Construction
35. Fragmentation,End Repair & A-tailing:
SECTION: 3’ Gene Expression Dual Index Library Construction
35.1. a)Prepare a thermal cycler with the following incubation protocol:

     Lid   Temperature      Reaction   Volume      Run time        65°C      50 ul      ~35 min        Step      Temperature      Time        Pre-cool   block      4°C      Hold        Fragmentation      32°C      0:05:00        End   Repair & A-tailing      65°C      0:30:00        Hold      4°C      Hold   

SECTION: 3’ Gene Expression Dual Index Library Construction
35.2. Vortex Fragmentation Buffer. Verify there is no precipitate.
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35.3. Prepare Fragmentation Mix on ice. Prepare Fragmentation Mix on ice. Pipette mix and centrifuge briefly. Pipette mix and centrifuge briefly.

     Reagent      1X (ul)      2X (ul)      3X (ul)      4X (ul)      5X (ul)      6X (ul)      7X (ul)      8X (ul)        Fragmentation Buffer      5      11      17      22      28      33      39      44        Fragmentation Buffer      10      22      33      44      55      66      77      88        Total      15      33      50      66      83      99      116      132   

SECTION: 3’ Gene Expression Dual Index Library Construction
35.4. Transfer ONLY 10 µl purified cDNA sample from Pellet Cleanup to a tube strip. The remaining 30 µl (75%) cDNA sample can be stored at 4°C for up to 72 h or at −20°C for up to 4 weeks for generating additional 3ʹ Gene Expression libraries.
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35.5. Add 25 µl Buffer EB to each sample.
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35.6. Add 15 µl Fragmentation Mix to each sample.
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35.7. Pipette mix 15x (pipette set to 35 µl) on ice. Centrifuge briefly.
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35.8. Transfer into the pre-cooled thermal cycler (4°C) and press “SKIP” to initiate the protocol.
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36. Post Fragmentation,End Repair & A-tailing Double Sided Size Selection – SPRIselect:
SECTION: 3’ Gene Expression Dual Index Library Construction
37. Adaptor Ligation:
SECTION: 3’ Gene Expression Dual Index Library Construction
36.1. Vortex to resuspend SPRIselect reagent. Add 30 µl SPRIselect (0.6X) reagent to each sample. Pipette mix 15x (pipette set to 75 µl)Vortex to resuspend SPRIselect reagent. Add 30 µl SPRIselect (0.6X) reagent to each sample. Pipette mix 15x (pipette set to 75 µl)
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36.2. Incubate 5 min at room temperature.
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36.3. Place on the magnet•High until the solution clears. DO NOT discard supernatant.
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36.4. Transfer 75 µl supernatant to a new tube strip.
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36.5. Vortex to resuspend SPRIselect reagent. Add 10 µl SPRIselect reagent (0.8X)to each transferred supernatant. Pipette mix 15x (pipette set to 80 µl).
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36.6. Incubate 5 min at room temperature.
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36.7. Place on the magnet•High until the solution clears.
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36.8. Remove 80 µl supernatant. DO NOT discard any beads.
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36.9. Add 125 µl 80% ethanol to the pellet. Wait 30 sec.
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36.11. Repeat steps 9 and 10 for a total of 2 washes.
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36.12. Centrifuge briefly. Place on the magnet•Low until the solution clears. Remove remaining ethanol.
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36.13. Remove from the magnet. Add 50.5 µl Buffer EB to each sample. Pipette mix 15x (pipette set to 45 µl).
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36.14. Incubate 2 min at room temperature.
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36.15. Place on the magnet•High until the solution clears.
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36.16. Transfer 50 µlsample to a new tube strip.
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36.10. Remove the ethanol.
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37.1. a)Prepare Adaptor Ligation Mix. Add reagents in the order listed. Pipette mix and centrifuge briefly.

     Reagent      1X (ul)      2X (ul)      3X (ul)      4X (ul)      5X (ul)      6X (ul)      7X (ul)      8X (ul)        Ligation Buffer      20      44      66      88      110      132      154      176        DNA Ligase      10      22      33      44      55      66      77      88        Adaptor Oligos      20      44      66      88      110      132      154      176        Total      50      110      165      220      275      330      385      440   

SECTION: 3’ Gene Expression Dual Index Library Construction
37.2. Add 50 µl Adaptor Ligation Mix to 50 µl sample. Pipette mix 15x (pipette set to 90 µl). Centrifuge briefly
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37.3. a)Incubate in a thermal cycler with the following protocol:

     Lid   Temperature      Reaction   Volume      Run time        30°C      100 ul      ~15 min        Step      Temperature      Time        1      20°C      0:15:00        2      4°C      Hold   

SECTION: 3’ Gene Expression Dual Index Library Construction
38. Post Ligation Cleanup – SPRIselect:
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38.1. Vortex to resuspend SPRIselect reagent. Add 80 µl SPRIselect (0.8X) reagent to each sample. Pipette mix 15x (pipette set to 75 µl).
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38.2. Incubate 5 min at room temperature.
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38.13. Transfer 30 µlsample to a new tube strip.
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38.3. Place on the magnet•High until the solution clears. 
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38.4. Remove the supernatant.
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38.5. Add 200 µl 80% ethanol to the pellet. Wait 30 sec.
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38.6. Remove the ethanol.
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38.7. Repeat steps 5 and 6 for a total of 2 washes.
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38.8. f)Centrifuge briefly. Place on the magnet•Low until the solution clears. 
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38.9. Remove remaining ethanol. Air dry for 2 min.
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38.10. Remove from the magnet. Add 30.5 µl Buffer EB to each sample. Pipette mix 15x.
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38.11. Incubate 2 min at room temperature.
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38.12. Place on the magnet•Low until the solution clears.
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39. Sample Index PCR:
SECTION: 3’ Gene Expression Dual Index Library Construction
39.1. Choose the appropriate sample index sets to ensure that no sample indices overlap in a multiplexed sequencing run. Record the 10x Sample Index name (PN-3000431 Dual Index Plate TT Set A well ID) used.
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39.2. Add 50 µl Amp Mix to 30 µl sample.
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39.3. Add 20 µl of an individual Dual Index TT Set A to each sample and record the well ID used. Pipette mix 5x (pipette set to 90 µl). Centrifuge briefly.
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39.4. a)Incubate in a thermal cycler with the following protocol:

     Lid   Temperature      Reaction   Volume      Run time        105°C      100 ul      ~ 25-40   min        Step      Temperature      Time        1      98°C      0:00:45        2      98°C      0:00:20        3      54°C      0:00:30        4      72°C      0:00:20        5      # Cycles   *        6      72°C      0:01:00        7      4°C      Hold   

* The total cycles should be optimized based on 25% carry forward cDNA yield/input calculated during cDNA Quantification
 
 
     Sequencing   Depth      Total   Cycles        Sequencing   Depth      14 - 16        Sequencing   Depth      12 - 14        Sequencing   Depth      10 - 12        Sequencing   Depth      8 - 10        Sequencing   Depth      6 - 8        Sequencing   Depth      5   

SECTION: 3’ Gene Expression Dual Index Library Construction
39.5. Store at 4°C for up to 72 h or proceed to the next step.
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40. Post Sample Index PCR Double Sided Size Selection – SPRIselect:
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40.1. Vortex to resuspend SPRIselect reagent. Add 60 µl SPRIselect (0.6X) reagent to each sample. Pipette mix 15x (pipette set to 150 µl).
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40.2. Incubate 5 min at room temperature.
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40.3. Place on the magnet•High until the solution clears. DO NOT discard supernatant.
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40.4. Transfer 150 µl supernatant to a new tube strip.
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40.5. Vortex to resuspend SPRIselect reagent. Add 20 µl SPRIselect reagent (0.8X)to each transferred supernatant. Pipette mix 15x (pipette set to 150 µl).
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40.6. Incubate 5 min at room temperature.
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40.7. Place on the magnet•High until the solution clears.
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40.8. Remove 165 µl supernatant. DO NOT discard any beads.
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40.9. Add 200 µl 80% ethanol to the pellet. Wait 30 sec.
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40.10. Remove the ethanol.
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40.11. Repeat steps 9 and 10 for a total of 2 washes.
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40.12. Centrifuge briefly. Place on the magnet•Low until the solution clears. Remove remaining ethanol.
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40.13. Remove from the magnet. Add 35.5 µl Buffer EB to each sample. Pipette mix 15x (pipette set to 35 µl).
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40.14. Incubate 2 min at room temperature.
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40.15. Place on the magnet•Low until the solution clears.
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40.16. Transfer 35 µlsample to a new tube strip.
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40.17. Store at 4°C for up to 72 h or at −20°C for long-term storage.
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41. Post Library Construction QC:
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41.1. Run 1 µl sample at 1:10 dilution on an Agilent Bioanalyzer High Sensitivity chip.
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41.2. Determine the average fragment size from the Bioanalyzer trace. This will be used as the insert size for library quantification.
SECTION: Sequencing
42. 3’ Gene Expression Library Sequencing Depth & Run Parameters:

     Sequencing Depth      Minimum 20,000 read pairs   per cell        Sequencing Type      Paired-end, dual indexing        Sequencing Read      Recommended Number of Cycles        Read 1      28 cycles            i7 index      10 cycles            i5 index      10 cycles            Read 2      90 cycles       

SECTION: Sequencing
43. Once quantified and normalized, the 3’ Gene Expression libraries should be denatured and diluted as recommended for Illumina sequencing platforms.