TITLE: UPitt TriState SenNet TMC Single Cell RNA Seq (10X Genomics) Library Preparation and Sequencing 5&#39; with Cell Hashings
AUTHORS: Tracy Tabib, Oliver Eickelberg, koenigshoffm, Robert Lafyatis

[DESCRIPTION]
This protocol is for the generation of 5' cDNA libraries from individual cells via droplet generation utilizing the 10x Genomics Chromium Controller and associated reagents. Paired-end libraries are then sequenced on Illumina instruments.
A modification of the "Chromium Next GEM Single Cell 5' Reagent Kits v2 (Dual Index)".

For Cell Hashing protocol:
"UPitt TriState SenNet TMC Cell Hashing of single cell suspension for scRNAseq in 5'; workflow (10x Genomics)"
dx.doi.org/10.17504/protocols.io.36wgqjdy5vk5/v1


[STEPS]
SECTION: GEM Generation &amp; Barcoding
1. Preparation of Single Cell Master Mix:
    a. Prepare Master Mix on ice. Pipette mix 15x and centrifuge briefly.


 


    b. Add 36.3 µl Master Mix into each tube of a PCR 8-tube strip on ice.
SECTION: GEM Generation &amp; Barcoding
2. Load Chromium Next GEM Chip K:
    a. Assemble Chromium Next GEM Chip
            Close the holder lid. 
            Attach the gasket by holding the tongue (curved end, to the right) and hook the gasket on the left-hand tabs of the holder. 
            Gently pull the gasket toward the right and hook it on the two right-hand tabs.
            Remove the chip from the sealed bag. Use the chip within ≤ 24 h
    b. Dispense 50% Glycerol Solution into each unused wells
(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
    c. Prepare Master Mix + Cell Suspension
            Refer to the Cell Suspension Volume Calculator Table 
            Add the appropriate volume of nuclease-free water to Master Mix.
            Add the appropriate volume of single cell suspension to Master Mix 
            (Total of 75 µl in each tube) 
            Gently pipette mix the single cell suspension before adding to the Master Mix.
    d. Load Row Labeled 1
            Gently pipette mix the Master Mix + Cell Suspension 
            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.
    e. Prepare Gel Beads
           • Snap the tube strip holder with the Gel Bead strip into a 10x Vortex Adapter. Vortex 30 sec.
           • Centrifuge the Gel Bead strip for ~5 sec.
           • Confirm there are no bubbles at the bottom of the tubes and the liquid levels are even.
           • Place the Gel Bead strip back in the holder. Secure the holder lid
    f. Load Row Labeled 2
           • Puncture the foil seal of the Gel Bead tubes.
           • Slowly aspirate 50 µl Gel Beads.
           • Dispense into the wells in row labeled 2 without introducing bubbles.
           • Wait 30 sec. 
    g. Load Row Labeled 3
           • Dispense 45 µl Partitioning Oil into the wells in row labeled 3 from a reagent reservoir.             Failure to add Partitioning Oil to the top row labeled 3 will prevent GEM generation and can damage the Chromium Controller
    h. Prepare for Run
            Close the lid (gasket already attached). DO NOT touch the smooth side of the gasket. DO NOT press down on the top of the gasket. 
             Run the chip in the Chromium Controller or X/iX immediately after loading the Partitioning Oil.
SECTION: GEM Generation &amp; Barcoding
3. Run the Chromium Controller
    a. Press the eject button on the Controller to eject the tray. 
    b. Place the assembled chip with the gasket in the tray, ensuring that the chip stays horizontal. Press the button to retract the tray. 
    c. Press the play button. 
    d. At the completion of the run (~18 min), the Controller will chime. Immediately proceed to the next step.

SECTION: GEM Generation &amp; Barcoding
4. Transfer GEMS: 
    a. Place a tube strip on ice. 
    b. Press the eject button of the Controller and remove the chip. 
    c. Discard the gasket. Open the chip holder. Fold the lid back until it clicks to expose the wells at 45 degrees. 
    d. Check the volume in rows labeled 1-2. Abnormally high volume in one well relative to other wells may indicate a clog. 
    e. 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 pipette tips and the bottom of the wells. 
    f. Withdraw pipette tips from the wells. GEMs should appear opaque and uniform across all channels. Excess Partitioning Oil (clear) in the pipette tips indicates a potential clog. 
    g. Over the course of ~20 sec, dispense GEMs into the tube strip on ice with the pipette tips against the sidewalls of the tubes. 
    h. 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 &amp; Barcoding
5. GEM-RT Incubation:
Use a thermal cycler that can accommodate at least 100 µl volume. A volume of 125 µl is the preferred setting on Bio-Rad C1000 Touch. In alternate thermal cyclers, use highest reaction volume setting. 
    a. Incubate in a thermal cycler with the following protocol. 
 

    b. 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
6. Post GEM-RT Cleanup & cDNA Amplification:
    a. 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.
            The resulting biphasic mixture contains Recovery Agent/Partitioning Oil (pink) and aqueous phase (clear), with no persisting emulsion (opaque).
            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. DO NOT invert without firmly securing the caps.
    b. Slowly remove and discard 125 µl Recovery Agent/Partitioning Oil (pink) from the bottom of the tube. DO NOT aspirate any aqueous sample. 
    c. Prepare Dynabeads Cleanup Mix.
 

    d. Vortex and add 200 µl to each sample. Pipette mix 10x (pipette set to 200 µl). 
    e. Incubate 10 min at room temperature (keep caps open). 
    f. Prepare Elution Solution I. Vortex and centrifuge briefly

 

    g. At the end of 10 min incubation, place on a 10x Magnetic Separator•High position (magnet•High) until the solution clears. A white interface between the aqueous phase and Recovery Agent is normal. 
    h. Remove the supernatant. 
    i. Add 300 µl 80% ethanol to the pellet while on the magnet. Wait 30 sec. 
    j. Remove the ethanol. 
    k. Add 200 µl 80% ethanol to pellet. Wait 30 sec. 
    l. Remove the ethanol. 
    m.Centrifuge briefly. Place on the 10x Magnetic Separator•Low position (magnet•Low). 
    n. Remove remaining ethanol. Air dry for 2 min. 
    o. Remove from the magnet. Immediately add 35.5 µl Elution Solution I. 
    p. Pipette mix (pipette set to 30 µl) without introducing bubbles. Pipette mix 15x. If beads still appear clumpy, continue pipette mixing until fully resuspended. 
    q. Incubate 1 min at room temperature. 
    r. Place on the magnet•Low until the solution clears. 
    s. Transfer 35 µl sample to a new tube strip. 
SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
7. cDNA Amplification: 
    a. Prepare cDNA Amplification Mix on ice. Vortex and centrifuge briefly.

 

    b. Add 65 µl cDNA Amplification Reaction Mix to 35 µl sample (Post GEM-RT Cleanup). 
    c. Pipette mix 5x (pipette set to 90 µl). Centrifuge briefly. 
    d. Incubate in a thermal cycler with the following protocol.

 

 

    e. Store at 4°C for up to 72 h or at −20°C for ≤1 week, or proceed to the next step.

SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
8. cDNA Cleanup – SPRIselect
    a. Vortex to resuspend the SPRIselect reagent. Add 60 µl SPRIselect reagent (0.6X) to each sample and pipette mix 15x (pipette set to 140 µl). 
    b. Incubate 5 min at room temperature. 
    c. Place on the magnet•High until the solution clears. 
    d. Transfer and save 80 µl supernatant in a new tube strip without disturbing the pellet. Maintain at room temperature. DO NOT discard the transferred supernatant (cleanup for Cell Surface Protein library construction). 
    e. Remove the remaining supernatant from the pellet without disturbing the pellet. DO NOT discard the pellet (cleanup for V(D)J and 5ʹGene Expression library construction). Immediately proceed to Pellet Cleanup.

SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
8.1. Pellet Cleanup (for V(D)J & 5ʹ Gene Expression library)
    i. Add 200 µl 80% ethanol to the pellet while still on magnet•High. Wait 30 sec. 
    ii. Remove the ethanol.
    iii. Repeat steps i and ii for a total of 2 washes. 
    iv. Centrifuge briefly and place on the magnet•Low. 
    v. Remove any remaining ethanol. Air dry for 2 min. DO NOT exceed 2 min as this will decrease elution efficiency. 
    vi. Remove from the magnet. Add 45.5 µl Buffer EB. Pipette mix 15x. 
    vii. Incubate 2 min at room temperature. 
    viii. Place the tube strip on the magnet•High until the solution clears. 
    ix. Transfer 45 µl sample to a new tube strip. 
    x. Store at 4°C for up to 72 h or at −20°C for up to 4 weeks, or proceed to cDNA QC & Quantification. 
SECTION: Post GEM-RT Cleanup &amp; cDNA Amplification
9. cDNA QC & Quantification
    a. Run 1 µl undiluted sample from the Pellet Cleanup step 2.3A-x (Dilution Factor 1) on an Agilent Bioanalyzer High Sensitivity chip. 
            Run 1 µl undiluted product for input cells with low RNA content (<1pg total RNA/cell), and 1 µl of 1:10 diluted product for input cells with high RNA content
    b. If proceeding to 5ʹ GEX Library Construction (step 5), determine cDNA yield for each sample.
SECTION: 5ʹ Gene Expression (GEX) Library Construction
10. 5ʹ Gene Expression (GEX) Library Construction
    a. Determine the volume for 50 ng mass of sample. Dispense the sample volume in a tube strip on ice. If the volume required for 50 ng is less than 20 µl, adjust the total volume of each sample to 20 µl with nuclease-free water. If the volume for 50 ng exceeds 20 µl, carry ONLY 20 µl sample into library construction. 
    b. Prepare a thermal cycler with the following incubation protocol. 
 
    c. Vortex Fragmentation Buffer. Verify there is no precipitate. 
    d. Prepare Fragmentation Mix on ice. Pipette mix and centrifuge briefly
 


    e. Add 30 µl Fragmentation Mix into each tube containing 20 µl sample. 
    f. Pipette mix 15x (pipette set to 30 µl) on ice. Centrifuge briefly. 
    g. Transfer into the pre-cooled thermal cycler (4°C) and press “SKIP” to initiate the protocol.
SECTION: 5ʹ Gene Expression (GEX) Library Construction
11. GEX Post Fragmentation, End Repair & A-tailing Double Sided Size Selection – SPRIselect
    a. Vortex to resuspend SPRIselect Reagent. Add 30 µl SPRIselect Reagent (0.6X) to each sample. Pipette mix 15x (pipette set to 75 µl). 
    b. Incubate 5 min at room temperature. 
    c. Place on the magnet•High until the solution clears. DO NOT discard supernatant. 
    d. Transfer 75 µl supernatant to a new tube strip. 
    e. Add 10 µl SPRIselect reagent (0.8X) to each sample. Pipette mix 15x (pipette set to 75 µl). 
    f. Incubate 5 min at room temperature. 
    g. Place on the magnet•High until the solution clears. 
    h. Remove 80 µl supernatant. DO NOT discard any beads. 
    i. With the tube strip still on the magnet, add 125 µl 80% ethanol to the pellet. Wait 30 sec. 
    j. Remove the ethanol. 
    k. Repeat steps i and j for a total of 2 washes. 
    l. Centrifuge briefly. Place on the magnet •Low. 
    m. Remove the ethanol. DO NOT over-dry beads to ensure maximum elution efficiency. 
    n. Remove from the magnet. Add 50.5 µl Buffer EB. Pipette mix 15x. 
    o. Incubate 2 min at room temperature. 
    p. Place on the magnet•High until the solution clears. 
    q. Transfer 50 µl sample to a new tube strip.
SECTION: 5ʹ Gene Expression (GEX) Library Construction
12. GEX Adaptor Ligation
    a. Prepare Adaptor Ligation Mix. Pipette mix and centrifuge briefly

 

    b. Add 50 µl Adaptor Ligation Mix to 50 µl sample. Pipette mix 15x (pipette set to 90 µl). Centrifuge briefly. 
    c. Incubate in a thermal cycler with the following protocol

 

SECTION: 5ʹ Gene Expression (GEX) Library Construction
13. GEX Post Ligation Cleanup – SPRIselect
    a. Vortex to resuspend SPRIselect Reagent. Add 80 µl SPRIselect Reagent (0.8X) to each sample. Pipette mix 15x (pipette set to 150 µl). 
    b. Incubate 5 min at room temperature. 
    c. Place on the magnet•High until the solution clears. 
    d. Remove the supernatant. 
    e. Add 200 µl 80% ethanol to the pellet. Wait 30 sec. 
    f. Remove the ethanol. 
    g. Repeat steps e and f for a total of 2 washes. 
    h. Centrifuge briefly. Place on the magnet•Low. 
    i. Remove any remaining ethanol. Air dry for 2 min. 
    j. Remove from the magnet. Add 30.5 µl Buffer EB. Pipette mix 15x. 
    k. Incubate 2 min at room temperature. 
    l. Place on the magnet•Low until the solution clears. 
    m. Transfer 30 µl sample to a new tube strip.
SECTION: 5ʹ Gene Expression (GEX) Library Construction
14. GEX Sample Index PCR
    a. 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. 
    b. Add 50 µl Amp Mix (PN-2000047/2000103) to 30 µl sample. 
    c. Add 20 µl of an individual Dual Index TT Set A to each well and record the well ID used. Pipette mix 5x (pipette set to 90 µl). Centrifuge briefly. 
    d. Incubate in a thermal cycler with the following protocol.

 

 
    e. Store at 4°C for up to 72 h or proceed to the next step.
SECTION: 5ʹ Gene Expression (GEX) Library Construction
15. GEX Post Sample Index PCR Double Sided Size Selection – SPRIselect
    a. Vortex to resuspend SPRIselect reagent. Add 60 µl SPRIselect reagent (0.6X) to each sample. Pipette mix 15x (pipette set to 150 µl). 
    b. Incubate 5 min at room temperature. 
    c. Place on the magnet•High until the solution clears. DO NOT discard supernatant. 
    d. Transfer 150 µl supernatant to a new tube strip. 
    e. Vortex to resuspend SPRIselect reagent. Add 20 µl SPRIselect reagent (0.8X) to each  sample. Pipette mix 15x (pipette set to 150 µl). 
    f. Incubate 5 min at room temperature. 
    g. Place on the magnet•High until the solution clears. 
    h. Remove 165 µl supernatant. DO NOT discard any beads. 
    i. With the tube strip still on the magnet, add 200 µl 80% ethanol to the pellet. Wait 30 sec. 
    j. Remove the ethanol. 
    k. Repeat steps i and j for a total of 2 washes. 
    l. Centrifuge briefly. Place on the magnet•Low. 
    m. Remove the remaining ethanol. DO NOT over-dry beads to ensure maximum elution efficiency. 
    n. Remove the tube strip from the magnet. Add 35.5 µl Buffer EB. Pipette mix 15x. 
    o. Incubate 2 min at room temperature. 
    p. Place on the magnet•Low until the solution clears. 
    q. Transfer 35 µl sample to a new tube strip. STOP 
    r. Store at 4°C for up to 72 h or at −20°C for long-term storage
SECTION: 5ʹ Gene Expression (GEX) Library Construction
16. GEX Post Library Construction QC
    a. Run 1 µl sample at 1:10 dilution on an Agilent Bioanalyzer High Sensitivity chip
    b. Determine the average fragment size from the trace. This will be used as the insert size for library quantification.
SECTION: Sequencing
20. Sequencing Libraries:
Chromium Single Cell 5' Gene Expression, and Cell Surface Protein Dual Index libraries comprise standard Illumina paired-end constructs which begin with P5 and end with P7. These libraries include 16 bp 10x Barcodes encoded at the start of TruSeq Read 1. Sample index sequences are incorporated as the i5 and i7 index read for 5' Gene Expression libraries; as i5 and i7 index read N for Cell Surface Protein library. 

SECTION: Cell Surface Protein Library Construction
17. Sample Index PCR
    a. 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-3000510 Dual Index Plate TN Set A well ID; verify name and part number) used. 
    b. Prepare Sample Index PCR Mix. 

 


    c. Transfer ONLY 5 µl sample from the Transferred Supernatant Cleanup to a new tube strip. 
Note that only 5 µl of the DNA sample is adequate for generating Cell Surface Protein library. The remaining DNA sample can be stored at 4°C for up to 72 h or at −20°C for up to 4 weeks for generating additional Cell Surface Protein libraries.
    d. Add 75 µl Sample Index PCR Mix to the 5 µl Transferred Supernatant Cleanup sample. 
    e. Add 20 µl of an individual sample index (Dual Index Plate TN Set A) to each well and record the well ID. Pipette mix 5x (pipette set to 90 µl). Centrifuge briefly. 
    f. Incubate in a thermal cycler with the following protocol.

 

SECTION: Sequencing
21. Sample Indices
Each well of the Dual Index Kit TT Set A (PN-1000215) and Dual Index Kit TN Set A (PN-1000250) contains a mix of one unique i7 and one unique i5 sample index. If multiple samples are pooled in a sequence lane, the sample index name (i.e. the Dual Index plate well ID) is needed in the sample sheet used for generating FASTQs with “cellranger mkfastq”. If multiple libraries are pooled in a sequence lane, a separate sample index is needed with each library
SECTION: Sequencing
22. Library Sequencing Depth & Run Parameters
    Sequencing Depth 
        Minimum 20,000 read pairs per cell for 5' Gene Expression Dual Index library 
        Minimum 5,000 read pairs per cell for Cell Surface Protein Dual Index library 
    Sequencing Type 
        Paired-end, Dual indexing 
    Sequencing Read 
        Read 1: 26 cycles 
        i7 Index: 10 cycles 
        i5 Index: 10 cycles 
        Read 2: 90 cycles
SECTION: Sequencing
23. Library Loading 
Once quantified and normalized, 5' Gene Expression, and Cell Surface Protein libraries should be denatured and diluted as recommended for Illumina sequencing platforms. Refer to Illumina documentation for denaturing and diluting libraries.

 

SECTION: Cell Surface Protein Library Construction
18. Post Sample Index PCR Size Selection – SPRIselect
    a. Vortex to resuspend the SPRIselect reagent. Add 120 µl SPRIselect Reagent (1.2X) to each sample. Pipette mix 15x (pipette set to 150 µl). 
    b. Incubate 5 min at room temperature.
    c. Place the magnet•High until the solution clears. Remove the supernatant. 
    d. Add 300 µl 80% ethanol to the pellet. Wait 30 sec. 
    e. Remove the ethanol. 
    f. Add 200 µl 80% ethanol to the pellet. Wait 30 sec. 
    g. Remove the ethanol. 
    h. Centrifuge briefly. Place on the magnet•Low. Remove remaining ethanol. 
    i. Remove from the magnet. Add 35.5 µl Buffer EB. Pipette mix 15x. 
    j. Incubate 2 min at room temperature. 
    k. Place on the magnet•Low until the solution clears. 
    l. Transfer 35 µl to a new tube strip. STOP 
    m. Store at 4°C for up to 72 h or at −20°C for long-term storage
SECTION: Cell Surface Protein Library Construction
19. Post Library Construction QC
    Run 1 µl sample at 1:10 dilution on an Agilent Bioanalyzer High Sensitivity chip.
    Determine the average fragment size from the Bioanalyzer trace. This will be used as the insert size for library quantification.