TITLE: FIND-seq protocol v1.0
AUTHORS: Iain C. Clark, Michael A. Wheeler, Hong-Gyun Lee, Liliana M. Sanmarco, Zhaorong Li, Shravan Thaploo, Carolina M. Polonio, Seung Won Shin, Giulia Scalisi, Joseph M. Rone, Federico Giovannoni, Stephanie E. J. Zandee, Alexandre Prat, Daniel C. Douek, Eli A. Boritz, Francisco J. Quintana, Adam R. Abate

[DESCRIPTION]
This protocol is a detailed description of FIND-seq, a single cell method for sorting cells based on RNA or DNA biomarkers. The protocol contains step-by-step instructions, key checkpoints, and troubleshooting guidelines. 

[GUIDELINES]
Equipment setup for microfluidic device operation
Detailed product information of the equipment used is given in the Materials tab.

Bright-field inverted microscope to visualize microfluidic device, droplet generation, re-injection, droplet sorting etc.
Four syringe pumps or equivalent pumps and Air-line with pressure gauge
Microtubing (PE2/PE5) to connect syringes to the device
Needles (23 gauge and 27.5 gauge), Dark green blunt tip needles (14 gauge, 2” tip, luer lock connection)
Sterile 1 mL syringes
Microfluidic devices; CAD files of the devices are provided on the Materials tab or on the Clark lab website (https://clarklab.berkeley.edu/protocols/) used for all experiments in the paper.



[STEPS]
SECTION: REAGENT SETUP
1. 20% (vol/vol) 1H-1H-2H-2H-Perfluoro-1-Octanol solution (Use this reagent immediately after preparation.)
 
      Reagent  Reagent conc.  Vol.    PFO  -  10 mL    HFE-7500  -  40 mL    Total    50 mL  
 
 

0.1% SPAN-80 in Hexane (Use this reagent immediately after preparation.)
      Reagent  Reagent conc.  Final conc.  Vol.    Span-80 (in hexane)  20% (w/v)  0.1 % (w/v)  0.25 mL    n-hexane  -  -  50 mL    Total      50.25 mL  
 

Cell Resuspension Buffer ( Solution is stable for several days at 4 °C.)
      Reagent  Reagent conc.  Final conc.  Vol.    HBSS buffer  1X  -  7.2 mL    Pluronic F-68  10% (v/v)  1% (v/v)  1 mL    Opti-prep  100%  18% (v/v)  1.8 mL    Total      10 mL  


Lysis Buffer (This solution is stable for several months at -20 °C and should be stored in small aliquots. When needed for experiment, add Proteinase K (2 µg/µL) to an aliquot and use immediately after preparation.)
      Reagent  Reagent conc.  Final conc.  Vol.    Tris-HCl pH 7.5  1000 mM  20 mM  0.2 mL    LiCl  8000 mM  1000 mM  1.25 mL    LiDS  10 % (v/v)  1 % (v/v)  1 mL    EDTA  500 mM  10 mM  0.2 mL    DTT  1000 mM  10 mM  0.1 mL    Proteinase K  20 µg/µL  2 µg/µL  1 mL    Nuclease free water  -  -  6.25 mL    Total      10 mL  
 

Wash 1 Buffer (This solution is stable for several months at 4 °C.) 
      Reagent  Reagent conc.  Final conc.  Vol.    Tris-HCl pH 7.5  1000 mM  20 mM  10 mL    LiCl  8000 mM  500 mM  31.25 mL    LiDS  10% (v/v)  0.1% (v/v)  5 mL    EDTA  500 mM  0.1 mM  0.1 mL    Nuclease free water  -  -  453.65 mL    Total      500  mL    Reagent  Reagent conc.  Final conc.  Vol.    Tris-HCl pH 7.5  1000 mM  20 mM  10 mL    NaCl  5000 mM  500 mM  50 mL    Nuclease free water  -  -  440 mL    Total      500 mL  
 

Wash 2 Buffer (This solution is stable for several months at 4 °C.)
      Reagent  Reagent conc.  Final conc.  Vol.    Tris-HCl pH 7.5  1000 mM  20 mM  10 mL    NaCl  5000 mM  500 mM  50 mL    Nuclease free water  -  -  440 mL    Total      500 mL  


5X RT Buffer (This solution is stable for several months at 4 °C.) 
      Reagent  Reagent conc.  Final conc.  Vol.    Tris-HCl pH 8.3  1000 mM  250 mM  50 mL    KCl  1000 mM  375 mM  75 mL    MgCl2  1000 mM  15 mM  3 mL    DTT  1000 mM  50 mM  10 mL    Nuclease free water  -  -  62 mL    Total      200 mL  


Tween Wash Buffer (This solution is stable for several months at room temperature.)
      Reagent  Reagent conc.  Final conc.  Vol.    Tween-20  100%  0.1% (v/v)  0.5 mL    Nuclease free water  -  -  500 mL    Total      500.5 mL  
 

Conjugation Buffer (Use this reagent immediately after preparation.) 
      Reagent  Reagent conc.  Final conc.  Vol.    Tris-HCl pH 8.3  1000 mM  375 mM  7.5 mL    Nuclease free water  -  -  12.5 mL  


10% (wt/vol) APS (Use this reagent immediately after preparation.)
 
      Reagent  Reagent conc.  Final conc.  Amount    APS  -  10% (w/v)  0.1 g    Conjugation buffer  -  -  upto 1 mL    Total      1 mL  

 
10% (vol/vol) TEMED (Use this reagent immediately after preparation.)
      Reagent  Reagent conc.  Final conc.  Vol.    TEMED  -  10% (v/v)  100 µL    Conjugation buffer  -  -  900 µL    Total      1000 µL  
(Caution: TEMED is toxic. Addition of TEMED should be done under a chemical fume hood. Wear appropriate protective clothing and equipment when handling it.)
SECTION: AGAROSE CONJUGATION
2. Prepare reagents for agarose conjugation. Resuspend Acrydite-T5-Smart-dT Primer in Conjugation Buffer to a concentration of 1000 micromolar (µM) (1 millimolar (mM) ). Resuspend SFR Allyl Agarose in Conjugation Buffer in a 15 mL falcon to a final concentration of 0.5 Mass / % volume. Prepare 10 Mass / % volume APS and 10 % (v/v) TEMED solutions.

SECTION: AGAROSE CONJUGATION
3. Heat SFR Agarose suspended in buffer to 95 °C for 120 min, or until completely molten. Vortex over time to ensure homogenization. While vortexing, it is good to flip the tube and vortex on head/cap as well. This ensures agarose near the top of the falcon does not cool and harden.

SECTION: AGAROSE CONJUGATION
4. Once homogenized, cool agarose to 45 °C. Temporarily placing heat block in ice is used to accomplish this faster.
SECTION: AGAROSE CONJUGATION
5. Place agarose under vacuum for 30 min. Ensure the agarose does not boil. If boiling is seen, agarose is not cool enough.
SECTION: AGAROSE CONJUGATION
6. Remove vacuum. Add reagents in the following order:
a. Add resuspended primer so final concentration of primer is 50 micromolar (µM).
b. Add 10 Mass / % volume APS solution so the final concentration is 0.1 Mass / % volume. Vortex tube thoroughly.
c. Add 10 % (v/v) TEMED solution so the final concentration is 0.1 % (v/v). Volume of APS and TEMED added should be the same. Vortex tube thoroughly.
SECTION: AGAROSE CONJUGATION
7. Place tube back under vacuum for 240 min. PAUSE POINT

SECTION: AGAROSE CONJUGATION
8. Remove tube from vacuum. Add the same volume of APS and TEMED as added in step 7. Final concentrations should now be 0.2 Mass / % volume  APS and 0.2 % (v/v)  TEMED. Vortex agarose and place under vacuum overnight at 45 °C. PAUSE POINT

SECTION: AGAROSE CONJUGATION
9. Remove agarose from vacuum. Heat to 95 °C and  vortex until completely molten and homogenized. Try to break polymer strands floating around by heating and vortexing as much as you can. Some strands may remain. While vortexing, it is good to flip the tube and vortex on the head/cap as well. This ensures agarose near the top of the tube does not cool and harden.

SECTION: AGAROSE CONJUGATION
10. While molten, pour agarose in a 10 mL syringe with a 0.45 µm syringe filter attached. Filter agarose into another 15 mL falcon tube to remove unwanted polymer strands formed during the reaction.
SECTION: AGAROSE CONJUGATION
11. Add ultra-low gelling temperature agarose to reach a final concentration of 2 Mass / % volume agarose (SFR and Ultra-low gelling together).

SECTION: AGAROSE CONJUGATION
12. After addition, heat tube to 95 °C and vortex to ensure homogenization, flipping tube on head occasionally.

SECTION: AGAROSE CONJUGATION
13. Once completely molten and homogenized, briefly centrifuge agarose to get all the agarose at the bottom of tube. Cool agarose to 4 °C by placing in ice bucket for at least 60 min. Allow agarose to harden.

SECTION: AGAROSE CONJUGATION
14. Using a hypodermic needle, carefully pierce the bottom of the falcon tube. This should dislodge the hardened agarose from the bottom of the tube. Wash by transferring hard agarose gel to a 500 mL bottle of nuclease free distilled water and allow to sit overnight. PAUSE POINT
SECTION: AGAROSE CONJUGATION
15. Repeat wash by re-transferring after the next night. Transfer agarose from wash bottle to a clean, dry 15 mL falcon tube. Ensure no water is transferred.
SECTION: DAY 1: CELL ENCAPSULATION AND LYSIS
18. Heat 3 mL of conjugated agarose-Oligo dT to 95 °C for at least 60 min. Vortex repeatedly to ensure homogenization and no solid clumps. CRITICAL STEP: Agarose must remain molten while running the device. Allowing agarose to cool down will clog the tubing/microfluidic channels.

SECTION: DAY 1: CELL ENCAPSULATION AND LYSIS
19. Prepare cells for encapsulation. It is recommended to start with at least 25-30 million cells. 
SECTION: DAY 1: CELL ENCAPSULATION AND LYSIS
20. Count cells using Trypan blue stain. For cell lines, you can use an automated cell counter (Countess II Automated Cell counter). For PBMCs, we recommend a manual hemocytometer.


SECTION: DAY 1: CELL ENCAPSULATION AND LYSIS
21. Based on the cell concentration, spin cells and resuspend in Cell Resuspension Buffer to 6.11x106 cells/mL.  We recommend resuspending in smaller volume of Opti-Prep than necessary to obtain a higher concentration, and then diluting down to the required concentration. 
*Troubleshooting
SECTION: DAY 1: CELL ENCAPSULATION AND LYSIS
22. Setup the drop making station
Keep the agarose molten and place the bubble triggered co-flow microfluidic device on the stage. Connect the syringe tubing to the microfluidic device. 
  

SECTION: DAY 1: CELL ENCAPSULATION AND LYSIS
23. Encapsulate cells in molten agarose using the bubble triggered co-flow microfluidic device at the following flow rates.

 

SECTION: DAY 1: CELL ENCAPSULATION AND LYSIS
24. Collect drops in 15 mL tubes in the heat block at 55 °C.

SECTION: DAY 1: CELL ENCAPSULATION AND LYSIS
25. Incubate for 120 min at 55 °C. Cool on ice or at 4 °C for at least 60 min or overnight. PAUSE POINT

SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
26. Remove oil and wash with hexane  (Caution: Hexane and PFO are highly flammable substances.)
Hardened agarose is henceforth referred to as beads. Remove oil at bottom of tube. Discard oil in appropriate waste bottle.
SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
27. Add 2x-5x volume 20 % volume HFE/PFO solution to drop emulsion. Mix slowly by inverting tube by hand to adequately break emulsion.

SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
28. Centrifuge at 2000 x g, 3 min. Oil will be at the bottom. Remove oil. Discard oil in waste bottle.

SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
29. Add >25 mL of Hexane/SPAN-80 solution to the beads. Shake tubes gently until beads are not clumpy. *Troubleshooting

SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
30. Centrifuge at 2000 x g, 3 min. Remove hexane with a pipette and discard in a hexane waste container. (CAUTION: Hexane damages plastics and should not be aspirated. Hexane must be disposed of in a glass waste bottle.) 
CRITICAL STEP: You must proceed through the reverse transcription reaction. Beads must be kept on ice at 4 °C throughout washes. Warming the beads will allow mRNA to dissociate from oligo-dT resulting in loss of mRNA and single cell resolution. 
SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
31. Water Washes
Add up to  50 mL of 4 °C Wash 1 Buffer. Resuspend by rotating by hand, flicking the bottom of the tube if necessary. Mix thoroughly and allow to sit on ice for 5 min. 
CRITICAL STEP: Residual hexane will be on top after first wash. Be careful to aspirate it and not let it mix in with the rest of the solution.

SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
32. Centrifuge for 4500 x g, 5 min. Aspirate buffer, not beads. The largest source of bead loss is getting too close to the water-agarose interface.  


SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
33. Add up to 50 mL Wash 2 Buffer. Resuspend by rotating by hand, flicking the bottom of the tube if necessary. Mix thoroughly and allow to sit on ice for 5 min.

SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
34. Pre-weigh a new 50 ml tube. Filter beads through 100 micron strainer into pre-weighted tube. *Troubleshooting
CRITICAL STEP: Pre-weighing the tube now is necessary for correctly setting up the reverse transcriptase reaction later in the protocol. Pre-weighed tube is used to calculate weight, and subsequently volume, of agarose beads left after washes.
SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
35. Centrifuge for 4500 x g, 5 min. Aspirate buffer, not beads.

SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
36. Add up to 50 mL Wash 2 Buffer. Resuspend by rotating by hand, flicking the bottom of the tube if necessary. Mix thoroughly and allow to sit on ice for 5 min.

SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
37. Centrifuge for 4500 x g, 5 min. Aspirate buffer, not beads
SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
38. Add up to 50 mL 5X RT Buffer. Resuspend by rotating by hand, flicking the bottom of the tube if necessary. Mix thoroughly and allow to sit on ice for 5 min.

SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
39. Repeat wash (steps 36 to 38) for a total of 2 RT Buffer washes.
SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
40. Reweigh tube after final aspiration to obtain weight of agarose beads. 
SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
41. Reverse Transcription Reaction
Beads should be in 5X RT Buffer on ice. Adding enzyme last, prepare reverse transcription reagents in a separate 50 mL falcon tube on ice as follows. Based on volume of washed beads, prepare reverse transcription mix.
 
      Reagent  Reagent conc.  Final conc.  Vol.    dNTP  25 mM  1 mM  1.2 mL    TSO  100 µM  2 µM  0.6 mL    MgCl2  1000 mM  6 mM  0.18 mL    Betaine  5 M  1 M  6 mL    PEG-8000  30% (w/v)  7.5% (w/v)  7.5 mL    Maxima H minus Reverse transcriptase  200 U/µL  2 U/µL  0.3 mL    NxGen Rnase inhibitor  20 U/µL  0.5 U/µL  0.75 mL    Nuclease free water  -  -  13.47 mL    Total      30 mL  
 

SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
42. Mix RT reaction using rotator for 30 min at room temperature and then 90 min at 42 °C on a rotator.  
SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
43. Take out of incubator, add 200 µL EDTA per 10 mL of reaction. Cool on ice for 10 min. PAUSE POINT

SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
44. Wash beads 5x in Tween Wash Buffer. On the last wash, filter with 100 micron strainer into a pre-weighed 50 mL Falcon tube.
SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
45. After the final wash with Tween Wash Buffer, weigh to determine the final mass of beads.
SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
46. Spin down beads for bead counting and take a 50 µL aliquot. PAUSE POINT
SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
47. Bead counting
Add 50 µL of beads to 150 µL of distilled nuclease free water to create a diluted bead stock.

SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
48. In a separate tube, mix a small aliquot of beads from the diluted bead stock with SYBR green (10x final concentration of dye). Let sit for 30 minin the dark.

SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
49. Using hemocytometer, image beads fluorescence microscope. Take pictures of bead size, bead counts, and pictures at 20x magnification of bead lysis success.  

SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
50. Quantify the number of beads/µL and genomes/µL. The ratio of genomes/bead should be ~1/10.
SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
51. Whole Transcriptome Amplification
Set up a 25 µL PCR reaction as follows, thermocycling using three different cycle numbers (14, 16, 18). PAUSE POINT 

      Reagent  Reagent conc.  Final conc.  Vol.    Kapa HiFi Master Mix  2 X  1 X  12.5 µL    SMART PCR Primer   10 µM  0.4 µM  1 µL    Beads    30 genomes/µL  X µL    Nuclease free water  -  -  11.5 -X µL    Total      25 µL  
 

 

SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
52. Use Ampure XP 2x beads for DNA clean-up. It is recommended to complete final elution in 20 µL- 30 µLof distilled nuclease free water. CRITICAL STEP: Over-drying of Ampure beads before elution step will result in loss of material.

SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
53. Measure DNA concentration using Qubit dsDNA kit. Based on Qubit results, take aliquots of sample and resuspend them to 1 ng/µL final concentration. Run these aliquots on a Bioanalyzer chip to confirm the size of WTA product.  *Troubleshooting

A good WTA product trace from Bioanalyzer looks like so:

  

SECTION: DAY 1: CELL ENCAPSULATION AND LYSIS
19.1. Preparing cells from cell lines in culture
Wash cells 1x in HBSS 400 x g, 3 min. Resuspend in 15 mL HBSS for each wash. Filter through a 70-micron strainer. 

SECTION: DAY 1: CELL ENCAPSULATION AND LYSIS
19.2. Preparing PMBCs
a. Place 25 mL of RPMI with 10 % volume FBS in 37 °C for 60 min.
b. Remove PBMC vial from freezer on dry ice. (CAUTION: Wear protective PPE, including a face shield, while defrosting PMBC vials.)
c. Submerge in 37 °C water bath until only a small amount of ice is visible
d. Pipette cells into media that was pre-warmed in 37 °C
e. Centrifuge cells for 300 x g, 10 min.
SECTION: DAY 2: BREAKING AND REVERSE TRANSCRIPTION
43.1. Alternatively, you can immediately begin bead washes with Tween Wash Buffer (next step).
SECTION: DAY 3: BEAD REINJECTION
54. Prepare a 2x PCR master mix.
Mix detection PCR reagents and beads in a 15mL falcon tube so that the final concentrations are:

      Reagent  Reagent conc.  Final conc.      TaqMan Probe  100 µM  250 nM  25 µL    TaqMan Forward Primer  100 µM  900 nM  90 µL    TaqMan Reverse Primer   100 µM  900 nM  90 µL    TaqPATH 2x Master Mix  2X  1x  5 mL    Tween-20  10% (v/v)  2.5% vol/vol  2.5 mL    PEG-6000  20% (w/v)  2.5% wt/vol  1.25 mL    Beads    -  X mL    Nuclease free water  -  -  1.045 - X mL    Total      10 mL  
 
SECTION: DAY 3: BEAD REINJECTION
55.  Soak for 60 min on shaker in the dark at room temperature.

SECTION: DAY 3: BEAD REINJECTION
56. Spin 15ml tubes at 4500 x g, 5 min to separate PCR mix and beads. Separate beads and PCR mix.  
Add PCR mix (20% of bead volume) to beads, vortex briefly. This prevents beads from aggregating in the device and makes reinjection stable. 
SECTION: DAY 3: BEAD REINJECTION
57. Load syringes 
Load PCR mix into a 3 mL syringe with a HFE oil backing (no fluorosurfactant)
Load beads into a 3 mL syringe
Load Droplet generation oil for Evagreen into a 3 mL syringe


SECTION: DAY 3: BEAD REINJECTION
58. Place PCR mix, beads, and oil into the syringe pumps and connect the tubing to the reinjection device.
  

SECTION: DAY 3: BEAD REINJECTION
59. Start the reinjection device with the following flow rates, to create ~70 µm diameter droplets:

 

SECTION: DAY 3: BEAD REINJECTION
60. Begin reinjection, collecting 30 µL aliquots into PCR strips. Occasionally during reinjection, verify that bead loading into droplets is close to 100% (or at least above 70%) by capturing videos.
SECTION: DAY 3: BEAD REINJECTION
61. After collection, thermocycle strips as follows with ramp rate set to 1.0 C/s

 


SECTION: DAY 4: SORTING DROPLETS
65. Flow rates for reinjecting into a detection/sorting device:

      Channel  Flow rate    Emulsion  100 µL/hr    Oil 1  400 µL/hr    Oil 2  2000 µL/hr    Oil 3  3000 µL/hr    Electrode  1000 V  
 
SECTION: DAY 4: SORTING DROPLETS
66. Determining cycle number for Amplification of sorted material
Cycling number is determined by performing WTA on a known number of sorted drops and calculating the number of cycles needed to obtain a desirable yield for positive drop sorts. 
SECTION: DAY 4: SORTING DROPLETS
67. For Single drop sorts
Collect single drops into PCR strips.
SECTION: DAY 4: SORTING DROPLETS
68. For 100 drop sorts
Collect drops in Eppendorf tubes.
SECTION: DAY 4: SORTING DROPLETS
68.1. Remove oil from Eppendorf tubes, leaving only a small amount to ensure that no unbroken drops are removed.
SECTION: DAY 4: SORTING DROPLETS
68.2. Add a 50 µL aqueous overlay (distilled nuclease-free water).

SECTION: DAY 4: SORTING DROPLETS
68.3. Spin tubes at 2000 rpm, 5 min. Then freeze tubes at -80 °C for at least 120 min or overnight. PAUSE POINT

SECTION: DAY 4: SORTING DROPLETS
68.4. Take tubes out of freezer. Heat tubes to 60 °C for 10 min. Remove samples from 60 °C and carefully mix only the aqueous layer by pipet.

SECTION: DAY 4: SORTING DROPLETS
68.5. Carefully transfer the aqueous layer in tubes to PCR strips. It is better to transfer some oil than to not transfer all aqueous.
SECTION: DAY 4: SORTING DROPLETS
68.6. Set up the PCR master mix as follows:
 
      Reagent  Reagent conc.  Vol. (for single tube)    KAPA buffer  5 X  10 µL    dNTP  10 mM  1.5 µL    PCR primer  100 µM  0.2 µL    Kapa   polymerase  1 µL    Nuclease   free water  0.3 µL  
 

 
SECTION: DAY 4: SORTING DROPLETS
67.1. After collection, overlay each tube with 37 µL of water.
SECTION: DAY 4: SORTING DROPLETS
67.2. Spin tubes at 20000 rpm, 5 min.

SECTION: DAY 4: SORTING DROPLETS
67.3. Freeze tubes at -80 °C for at least 120 min or overnight. PAUSE POINT
SECTION: DAY 4: SORTING DROPLETS
67.4. Take tubes out of freezer and heat tubes to 60 °Cfor 10 min.
SECTION: DAY 4: SORTING DROPLETS
67.5. Using the table below, prepare a Master mix.

 
      Reagent  Reagent conc.  Vol. (for single tube)    KAPA buffer  5 X  10 µL    dNTP  10 mM  1.5 µL    PCR primer  100 µM  0.2 µL    Kapa   polymerase  1 µL    Nuclease   free water  0.3 µL  
 

 
 
SECTION: DAY 4: SORTING DROPLETS
67.6. Add 13 µL of PCR reagents for 50 µL total reaction volume.
SECTION: DAY 4: SORTING DROPLETS
67.7. Flick the tube to mix, spin, and thermocycle.
SECTION: DAY 4: SORTING DROPLETS
67.8. Thermocycle according to previously determined cycle number (Step 53).
SECTION: DAY 4: SORTING DROPLETS
67.9. Cleanup WTA with 1.2X Ampure XP.
Add 60 µL Ampure XP beads to 50 µL WTA reaction.
SECTION: DAY 4: SORTING DROPLETS
68.7. Add 17 µL of PCR master mix to 50 µL of aqueous layer

SECTION: DAY 4: SORTING DROPLETS
68.8. Carefully mix by flicking the PCR tubes. Do not form emulsions. After mixing, spin tubes to separate aqueous and oil layers.
SECTION: DAY 4: SORTING DROPLETS
68.9. Thermocycle according to previously determined cycle number (Step 53).
SECTION: DAY 4: SORTING DROPLETS
68.10. Cleanup WTA with 1.2X Ampure.
Add 80.4 µL Ampure XP beads to 67 µL WTA reaction.

SECTION: Troubleshooting
69.  
     Step      Problem      Possible Reason      Solution        19.      Cells are all dead/Counts seem incorrect.      ●    Automated Cell counter may not   be able to differentiate between live and dead cells for smaller cell types.   ●    Cells may not have been mixed   thoroughly      Mix cell resuspension thoroughly and count cells using a   hemocytometer slide.        27      Solution seems to have solidified/beads are not   separating from hexane      ●    Issue with agarose concentration  ●    Generated double emulsions   during addition of HFE/PFO in step 25      Restart Experiment        32      Beads not passing through the filter/Film of beads on filter   leading to large loss      ●    Beads are larger than normal   (>55 µm) and struggle to pass through 100 µm filter.   ●    Large amount of beads floating   around in solution      Split original tube from which beads came from into   multiple tubes. Use multiple filters if necessary, adding WASH BUFFER 2 to   help correctly sized beads pass through the filter. If tubes are split, it is   best to recombine tubes while beads are suspended in any of the wash buffers.        51      Yield of DNA material obtained after WTA reaction is low/zero      ●    Low/zero yield may be due to   failure of reverse transcription reaction  ●    Low/zero yield may be due to   mRNA loss during initial washes before reverse transcriptase reaction. This   happens when buffers used are not high enough salt concentration or when   beads are not kept on ice and mRNA dissociates from the poly-T tail within   conjugated agarose beads.       Restart Experiment   
 
SECTION: DAY 4: SORTING DROPLETS
63. Load syringes 
Load thermocycled emulsion into a 3 mL syringe
Load Droplet generation oil for Evagreen into a 3 mL syringes (Oil 1)
Load HFE-7500 into a 3 mL syringes (Oil 2)
Load HFE-7500 into a 3 mL syringes (Oil 3)

SECTION: Agarose Normalization
17. Normalize the agarose concentration
SECTION: Agarose Normalization
16.4. Calculate the average µM concentration of conjugate oligonucleotide. Use the reported molecular weight on the IDT datasheet for the oligo-dT primer. Remember to consider the 80-fold dilution. 

e.g. If I get 5.52 ng/µL Qubit reading (1/80 dilution) for a 30 base ssDNA primer:  
Molecular concentration (µM) = Qubit reading (ng/µL) * Dilution factor (80) * 1000 / Molecular weight of total primer (g/mol)  
Molecular concentration (µM) = 5.52 ng/ul * 80 * 1000 / 9378.2 g/mol = 39 µM
SECTION: Agarose Normalization
16.3. In triplicate, measure conjugated oligo-dT via the QuBit ssDNA Assay kit.
SECTION: Agarose Normalization
16.2. Dilute agarose in nuclease free water (1/80 dilution). Vortex to ensure agarose added is dissolved and evenly distribute. Triplicates are ready for measurement.
SECTION: Agarose Normalization
16.1. Melt agarose at 95 °C. Vortex and homogenize. Once agarose is molten, cool down to 70 °C or until viscosity is amenable to pipetting. Use ice to cool agarose tube down.

SECTION: Agarose Normalization
16. Quantify the oligonucleotide concentration
SECTION: Agarose Normalization
17.1. Normalize the conjugated oligonucleotide concentration by addition of 2 Mass / % volume Ultra-low melt Agarose. Prepare 2 Mass / % volume Ultra-low melt Agarose by adding 0.08 g of agarose to 4 mL of distilled nuclease free water. Vortex and heat to 95 °C. Keep vortexing until completely dissolved.
SECTION: Agarose Normalization
17.2. Dilute conjugated agarose in 2 Mass / % volume Ultra-low melt Agarose to reach a final concentration of 8 micromolar (µM) conjugated oligonucleotide.

SECTION: Agarose Normalization
17.3. Measure final conjugated oligonucleotide concentration with QuBit ssDNA kit using the same method as above. The final concentration should be 8 micromolar (µM).
SECTION: DAY 4: SORTING DROPLETS
62. See previously published paper of Mazutis et. al (Nat. Protoc. 2013 May;8(5):870-91.) for details on droplet sorting procedure.
SECTION: DAY 4: SORTING DROPLETS
66.1. Sort 6-9 aliquots of the number of cells (# cells) that you will be collecting in the real experiment. 
SECTION: DAY 4: SORTING DROPLETS
66.2. Perform WTA and bioanalyzer using 3 different cycle numbers in duplicate (or triplicate)
SECTION: DAY 4: SORTING DROPLETS
66.3. Plot the data and select the number of cycles that gives at least 2 ng/µL in 20 µL elution volume, or 40 ng of DNA, determined by qubit or bioanalyzer.

SECTION: DAY 4: SORTING DROPLETS
64. Place emulsion, and oil into the syringe pumps and connect the tubing to the sorting device. Fill the saltwater electrode and moat channels with 2 M NaCl solution. Connect saltwater electrode to voltage amplifier.