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InTraSeq™ 3' Protocol

InTraSeq™ 3' Protocol

For best results:

  • Maintain an RNase-free and sterile workspace.
  • It is highly recommended to use filtered pipette tips.
  • When starting Section B. Blocking (Day 2) until the end of the protocol, it is crucial to perform the centrifugations at 850 x g (not rpm).
  • When starting Section C. Immunostaining (Day 2), it is crucial to maintain the cell pellet submerged (in ~40 µL) when removing and discarding the supernatant.
  • Do not vacuum aspirate when discarding the supernatant in any step; always use a pipette.
  • Make sure the reagents are completely thawed before use.

NOTES:

  • All InTraSeq™ 3' Assay Kit components are validated by CST in the 10x Genomics Chromium Single Cell 3' Kits with Feature Barcoding technology.
  • SAFE STOP - This indicates a safe stopping point in the protocol if stopping is necessary.
  • This protocol is designed for a single InTraSeq™ assay (one sample). If processing multiple samples, all quantities should be adjusted proportionally.

Solutions and Reagents

Materials Included in Kit (Store at -20°C)

  • InTraSeq™ Blocking Buffer A #10471
  • InTraSeq™ Blocking Buffer B #31701
  • InTraSeq™ Staining Buffer #46005
  • InTraSeq™ Wash Buffer #59335
  • InTraSeq™ Reducing Agent #63760
  • InTraSeq™ RNase Inhibitor #79298
  • Rabbit (DA1E) Monoclonal Antibody IgG Isotype Control (InTraSeq™ 3' Conjugate 3000) #81472
  • Histone H3 (D1H2) Rabbit Monoclonal Antibody (InTraSeq™ 3' Conjugate 3002) #68984

Required Reagents Not Included

  • InTraSeq™ 3' Conjugates/Antibody Cocktails
  • Two sterile 40-micron Falcon cell strainers
  • Vortex mixer
  • 1.5 mL Sterile Polypropylene Microcentrifuge Tubes - LoBind
  • 15 mL Conical Sterile Polypropylene Centrifuge Tubes (preferably LoBind)
  • 50 mL Conical Sterile Polypropylene Centrifuge Tubes (preferably LoBind)
  • Phosphate Buffered Saline (PBS-1X) pH7.2 (Sterile) #9872
  • Methanol #13604
  • Human Fc Receptor Blocking Solution #58948 (if using G4S Linker (E7O2V) Rabbit Monoclonal Antibody (InTraSeq™ 3' Conjugate 3118) #38470)
  • Antibody Dilution Buffer #13616 (if using G4S Linker (E7O2V) Rabbit Monoclonal Antibody (InTraSeq™ 3' Conjugate 3118) #38470)

Before starting:

(Optional) When you receive the kit, thaw and pipette mix the InTraSeq™ Blocking Buffer A, then aliquot 875 µL into 8 separate 1.5 mL sterile microcentrifuge tubes.

A. Cell Fixation (Day 1)

  1. Count and transfer 1-5 million cells to a 15 mL tube; alternatively, count and transfer at least 2-5 million cells if performing an immunostaining step using G4S Linker (E7O2V) Rabbit Monoclonal Antibody (InTraSeq™ 3' Conjugate 3118) #38470.

    NOTE: Do not proceed with the protocol with less than 1 million cells.

  2. Centrifuge the cells at 300 x g for 5 min at 4°C.
  3. Remove and discard the supernatant, then wash the cells with 10 mL of ice-cold 1X PBS.
  4. Centrifuge the cells at 300 x g for 5 min at 4°C.
  5. Proceed to Step 9; alternatively, if performing an immunostaining step using G4S Linker (E7O2V) Rabbit mAb (InTraSeq™ 3' Conjugate 3118) #38470, proceed to Step 6.
  6. Treat 2-5 million cells with Human Fc Receptor Blocking Solution #58948 for 10 min at room temperature, using 2.5 µg/1 million cells in ~100 µL Antibody Dilution Buffer #13616 (or a solution of 0.5% Bovine Serum Albumin in 1X Phosphate Buffered Saline).
  7. Add 3 µL G4S Linker (E7O2V) Rabbit Monoclonal Antibody (InTraSeq™ 3' Conjugate 3118) #38470 and incubate at 4°C for 30 min.
  8. Add 2 mL of Antibody Dilution Buffer #13616 to the cells (or a solution of 0.5% Bovine Serum Albumin in 1X Phosphate Buffered Saline). Pipette mix to resuspend the cells.
  9. Centrifuge at 300 x g for 5 min at 4°C, then remove and discard the supernatant without disturbing the cell pellet.
  10. Repeat steps 8 and 9 twice for a total of 3 washes.
  11. Resuspend the cells in 0.5 mL of ice-cold 1X PBS.
  12. Turn on the vortex mixer and keep it on the “On” switch (not the “Auto/Touch” mode) at low speeds (~speed 1-4 depending on your mixer). The sample should NOT be vortexed, but it should rather be constantly mixed. If the mixer is fast enough to vortex the sample, decrease the speed accordingly.

    NOTE: It is very important to drop the ice-cold methanol slowly (> 30 sec) in step 13. below.

  13. Ensure that the vortex mixer is on the "On" switch, place the 15 mL tube on the vortex mixer, then slowly add 4.5 mL of ice-cold methanol in a dropwise manner (> 30 sec).
  14. Incubate the cells overnight in the -20°C freezer. (SAFE STOP - SAMPLE CAN BE STORED FOR UP TO 7 d IN THE FREEZER AT -20°C OR UP TO 14 d AT -80°C)

B. Blocking (Day 2)

From this point forward, at every centrifugation step, it is crucial to spin down the cells at 850 x g. Decreasing the spin down speed will result in cell loss.

  1. Thaw the following reagents at room temperature:
    • Thaw 1 bottle of InTraSeq™ Wash Buffer.
    • Thaw the InTraSeq™ Blocking Buffer B.
    • Thaw and pipette mix the InTraSeq™ Blocking Buffer A.
    • Thaw and pipette mix the InTraSeq™ Staining Buffer.
    • Thaw and vortex mix InTraSeq™ Reducing Agent until the precipitate disappears.
  2. Once thawed, place the reagents on ice.
  3. Prepare the heat/cooled InTraSeq™ Blocking Buffer B:
    1. Aliquot 130 µL InTraSeq™ Blocking Buffer B into a sterile 1.5 mL microcentrifuge tube. Return the stock InTraSeq™ Blocking Buffer B to the -20°C freezer.
    2. Heat the aliquoted tube at 95°C for 5 min.
    3. Spin the tube down briefly, then place it on ice. Use this heat/cooled InTraSeq™ Blocking Buffer B in the steps below.
  4. Prepare the scBlock (see table below), pipette mix. Store on ice.
    scBlock Volume
    InTraSeq™ Blocking Buffer A 860 µL
    Heat/cooled InTraSeq™ Blocking Buffer B 98 µL
    InTraSeq™ Reducing Agent 40 µL
    InTraSeq™ RNase Inhibitor 2 µL
    TOTAL Volume 1,000 µL
  5. Centrifuge the cells at 850 x g for 5 min at 4°C.
  6. Remove and discard the supernatant, then add the scBlock to the cells. Pipette mix to resuspend the cells, then keep the cells on ice for at least 30 min.
  7. During the scBlock incubation, prepare the Antibody Master Mix as follows:
    1. Determine the volume of Antibodies (Vol. of Abs.) based on the number of InTraSeq™ 3' Antibody Cocktails and Individual InTraSeq™ 3' Conjugates (Ind. InTraSeq™ 3' Conj. or Ind. Conj.) being used (see table below). Limit the total volume to 30 µL.
        Volume
      (Cocktails only)      		 Volume
      (Ind. Conj. only)      		 Volume
      (Cocktails + Ind. Conj.)
      InTraSeq™ 3' Cocktail  5 µL (per cocktail) N/A ___ µL (5 µL per cocktail)
      Ind. InTraSeq™ 3' Conj. N/A 3 µL (per Ind. Conj.) ___ µL (3 µL per Ind. Conj.)
      Vol. of Abs. ___ µL ___ µL ___ µL
    2. Prepare the Antibody Master Mix (see table below) using the Vol. of Abs. (max 30 µL) calculated above. Gently pipette mix to minimize bubble formation, then store on ice.
      Antibody Master Mix Volume
      (Cocktails only)      		 Volume
      (Ind. Conj. only)      		 Volume
      (Cocktails + Ind. Conj.)
      Vol. of Abs. ___ µL ___ µL ___ µL
      InTraSeq™ Wash Buffer 30 µL - Vol. of Abs. (see row above)
      InTraSeq™ Staining Buffer 55 µL 55 µL 55 µL
      Heat/cooled InTraSeq™ Blocking Buffer B 10 µL 10 µL 10 µL
      Rabbit (DA1E) Monoclonal Antibody IgG Isotype Control (InTraSeq™ 3' Conjugate 3000) 3 µL 3 µL 3 µL
      Histone H3 (D1H2) Rabbit Monoclonal Antibody (InTraSeq™ 3' Conjugate 3002) or any other Histone H3 (D1H2) Rabbit Monoclonal Antibody (InTraSeq™ 3' Conjugate) 3 µL 3 µL 3 µL
      InTraSeq™ RNase Inhibitor 2 µL 2 µL 2 µL
      TOTAL Volume 103 µL 103 µL 103 µL

    8. NOTE: If multiplexing multiple InTraSeq samples with Histone H3 (D1H2) InTraSeq 3' Conjugates, count and aliquot the same number of cells from each sample before starting step 8.

  8. After the 30 min scBlock incubation, add 2 mL InTraSeq™ Wash Buffer to the cells and pipette mix.
  9. Filter the cells through a sterile 40-micron Falcon cell strainer into a 50 mL tube.

    NOTE: To maximize cell recovery, apply gentle pressure to the pipette tip against the strainer while filtering. (See image)

    Image 1
  10. Transfer the flow through/filtered cells into a new 15 mL tube and immediately start Section C. Immunostaining (Day 2).

    NOTE: Store the remaining InTraSeq™ Wash Buffer at 4°C for use on Day 3.

C. Immunostaining (Day 2)

From this point forward, whenever removing and discarding the supernatant, it is crucial to maintain the cell pellet submerged (in ~40 µL). Drying the cells will negatively impact the RNA signal and result in cell loss.

Before starting:

  1. Centrifuge the cells at 850 x g for 5 min at 4°C.
  2. Remove and discard supernatant while retaining ~40 µL in the tube.
  3. Add 100 µL Antibody Master Mix to the cells. Pipette mix to resuspend the cells.
  4. Transfer to a sterile 1.5 mL LoBind microcentrifuge tube and incubate at 4°C overnight (for ~16 hr).

    NOTES:

    • Do not incubate the cells for longer than 20 hr.
    • It is crucial to maintain the overnight incubation at 4°C.

D. Washes and Count (Day 3)

Before starting:

  • Prepare the Modified InTraSeq™ Wash Buffer. Gently mix to minimize bubble formation, then keep on ice.

    NOTE: If there is remaining InTraSeq™ Wash Buffer in the stock bottle, store it at -20°C.

    • Image 2
  1. Add 1 mL Modified InTraSeq™ Wash Buffer to the 1.5 mL microcentrifuge tube containing the cells. Pipette mix, then transfer the mixture to a 15 mL tube.
  2. Add 2 mL Modified InTraSeq™ Wash Buffer so the total volume is 3 mL.
  3. Centrifuge at 850 x g for 5 min at 4°C. Remove and discard supernatant while retaining ~40 µL in the tube.
  4. Add 1 mL Modified InTraSeq™ Wash Buffer. Pipette mix to resuspend the cells, then add another 3 mL Modified InTraSeq™ Wash Buffer so the total volume is 4 mL.
  5. Repeat Steps 3 and 4, then filter the 4 mL cell suspension through a sterile 40-micron Falcon cell strainer into a 50 mL tube. Transfer the flow through/filtered cells into a new 15 mL tube.

    NOTE: To maximize cell recovery, apply gentle pressure to the pipette tip against the strainer while filtering. (See image)

    Image 1
  6. Centrifuge the cells at 850 x g for 5 min at 4°C. Remove and discard supernatant while retaining ~40 µL in the tube.
  7. Add 100 µL Modified InTraSeq™ Wash Buffer to the cells. Pipette mix to resuspend the cells, then keep the cells on ice.

    NOTE: From this point forward, keep the cells in the Modified InTraSeq™ Wash Buffer, including any necessary dilutions. Using any other buffer or solution will result in RNA degradation.

  8. Count the cells. Refer to the Cell Suspension Volume Calculator Table in the 10x Genomics Chromium Single Cell 3' Kits with Feature Barcoding technology protocol to determine the desired cell concentration.

    IMPORTANT: Dilute the cells using ONLY the Modified InTraSeq™ Wash Buffer to reach the desired cell concentration.

    NOTE: If cell clumps are observed after counting, additional pipette mixing is recommended before proceeding to the single-cell experiment.

  9. Process the cells using the 10x Genomics Chromium Single Cell 3' Kits with Feature Barcoding technology.

For Research Use Only. Not for Use in Diagnostic Procedures.