inDrop Single Cell Encapsulation and Reverse Transcription ...€¦ · 1X Phosphate Buffered Saline VWR Scientific Cat. No. 12001-676 Trypan Blue (0.4%) Thermo Fisher Cat. No. T10282

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Single Cell Encapsulation Protocol, Version 2.4

inDrop™ Single Cell Encapsulation

and Reverse Transcription Protocol, Version 2.4

Table of Contents

A. Reagents needed for the inDrop™ Single Cell Reverse Transcription B. RNase-free Sample Handling C. Cell Fitness D. Step-by-Step Protocol for Barcoding Cells

1. Instrument Setup 2. Hydrogel Bead Preparation 3. Cell and RT/Lysis Buffer Preparation 4. Loading the Microchip 5. Initiating Gel Bead Encapsulation 6. Optimizing Gel Bead Encapsulation 7. cDNA Synthesis 8. Instrument Shutdown

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Single Cell Encapsulation Protocol, Version 2.4 2

A. Reagents and Equipment for the inDrop™ Single Cell RT

Provided by 1CellBio in the inDrop scRNA-seq kit

Storage at 4°C o Barcoded Hydrogel Microspheres (BHMs) [500,000 BHMs are provided in 500 µl]

Storage at -20°C o 1.3x RT Premix

o 2x Gel Concentration buffer

Storage at Room Temperature o HFE 7500

o Wash Buffer

o OptiPrep - Density-matching Agent

o Droplet-making Oil

o Demulsifying Agent

o 1 M MgCl2

o Mineral Oil

o Silane

o Storage Buffer

o 1 mL syringes

o PTFE filters

o 2 Colored oil syringe assemblies

o Syringe needles

o 3 mL syringe

o Collection tubing

o Microfluidics Chip

o Colored Oil

o Pipette tip assemblies

o Droplet oil syringe assembly

o BHM loading assembly

Reagents and Equipment not provided by 1Cell Bio SuperScript III Reverse Transcriptase Thermo Fisher Cat. No. 18080044

RNaseOUT Recombinant Ribonuclease Inhibitor Thermo Fisher Cat. No. 10777019

DTT Provided in SuperScript III kit

1X Phosphate Buffered Saline VWR Scientific Cat. No. 12001-676

Trypan Blue (0.4%) Thermo Fisher Cat. No. T10282

1.5 mL LoBind tube Eppendorf AG, Cat. No. 022431021

Thermo-conductive tube rack VWR Scientific Cat. No. 95045-464

UV Lamp or 1CellBio UV Device VWR Scientific Cat. No. 36595-020

Heat block for 1.5 mL tubes VWR Scientific Cat. No. 12621-084

Aluminum foil Your favorite supplier

Razor blades or scalpel Your favorite supplier

0.45 μm syringe filter CellTreat Product Num. 229758

Vortex Your favorite supplier

Centrifuge Eppendorf AG, Cat. No. 5424R

Hemocytometer and cover slips Your favorite supplier

Gel loading pipet tips VWR Scientific Cat. No. 37001-152

Tweezers Your favorite supplier

Lens wipes Parafilm

VWR Scientific Cat. No. 52846-001

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B. RNase-free Sample Handling

1CellBio recommends using proper sterile technique, including RNase-free precautions, when performing single cell transcriptome processing on any sample.

o Wear gloves at all times. Wear gloves not only when handling samples directly but also when handling any equipment or reagents. Change gloves often particularly after touching skin or hair.

o Clean your workspace and equipment with commercially available products

designed to remove RNases. Wipe down all work surfaces, pipettes, racks, centrifuges, PCR machines and other equipment to be used during or in the vicinity of your sample processing. Ideally keep a set of clean pipettes dedicated exclusively to RNA work in your lab.

o Use RNase-free reagents.

Always use sterile tubes and reagents labeled as DNase and RNase free. Use aerosol-free disposable pipette tips, nuclease-free water and nuclease-free sample centrifuge tubes. Always shake tubes you need onto a clean surface rather than reaching a hand into the bag.

o Keep reagents clean.

If you require small amounts of a reagent that is supplied in a large volume create a working stock of a smaller volume in a sterile tube and discard the excess at the end of each day or if you are concerned the aliquot has become contaminated. Only access reagents using sterile pipette tips. Never breach a reagent tube or bottle with the shaft of your pipette.

Note – Following the few guidelines above will help insure successful processing of your single cell transcriptome samples. If you are unfamiliar with general sterile technique, are inexperienced working with RNA or would like additional recommendations for proper processing of RNA samples there are a wide variety of resources available on the internet we encourage you to review before proceeding.

C. Cell Fitness Prior to running the protocol, use a hemocytometer to confirm your cell sample meets the following requirements:

o The cell suspension has very few, or no, cell doublets or clumps (less than 5%). Cell

strainers may be used to filter out cell clumps.

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o The sample is free from cell lysate (as lysates may contain RNA which may contaminate

the sample).

o Cell viability in 1x PBS is high and remains stable for at least 30 min on ice. Ideally, the

viability of the sample will be above 95% and will remain above 90% after 30 minutes on

ice.

D. Step-by-Step Protocol for Barcoding Cells

1. Instrument Setup

a) Log into the laptop provided by 1CellBio – the login password is “inDrop” and Click desktop icon labeled “Thor”. This opens the software that controls the instrument and implements the inDrop process.

b) Remove the BHM loading assembly from its designated package and mount it on the pump marked “Hydrogel Beads”. To avoid tubing priming on the instrument, begin to hand prime the syringe by pushing on the plunger for 200ul with the syringe pointing upwards. Note – the syringe is shipped pre-filled with hydraulic fluid specific to the loading the hydrogel beads. If the amount of fluid becomes depleted, refill the syringe with 1:1 mixture of Wash Buffer and 2x Gel Concentration Buffer.

c) Remove the syringe assembly and mount the designated droplet making oil syringe on to the pump marked “Droplet Oil”. Prime the tubing by using Thor and infusing the Droplet Making Oil from the syringe, into the tubing. Once a drop forms at the tip of the tubing, stop the pump and remove the excess fluid with a low lint lens wipe.

d) Remove the colored oil filled syringe assembly marked “Cells” from the bag and mount the assembly on the corresponding pump.

e) Repeat steps e for the syringe pump marked “RT Mix”.

f) On the first tab of Thor, “Set Up”, input the; total number of cells to encapsulate, the

concentration of the cell solution, and the Bead-to-cell ratio desired. Then press “Calculate”.

g) This will populate the boxes below with the amount, in microliters, of reagents needed

to be prepped. The “?” gives the recipe of the RT/Lysis Mixture to be made.

h) Prepare microchip devices by following the “Chip Silanizing” protocol that can be

downloaded through http://www.1cell-bio.com/.

i) When the beads are ready to be prepared, press “Next”

Critical: Samples must be processed at no more than 3,000 cells at a time to minimize repeating a barcode. The samples can be collected separately or the emulsion can be divided post reverse transcription reaction. For experiments over 6,000 cells or 20 mins, it is recommended to reload. fresh RT Mix.

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j) If planning on doing multiple experiments, calculate bead volume for the total number

of cells desired. Load BHMs according to this amount. When moving to new sample, recalculate other volumes specifically for that experiment.

2. Hydrogel Bead Preparation

a) In dim lighting, remove tube of BHM from provided box.

b) Concentrate the barcoded hydrogel microspheres (BHMs) in the stock, 1.5 mL tube by

spinning at 5000 g for 2 min. Carefully examine the tube to be sure you can distinguish

the bead pellet from the supernatant.

c) Carefully transfer 250 µL of the concentrated BHMs to a new 1.5 mL LoBind Eppendorf

tube and begin the BHM washes. Note – 250 µL is sufficient for barcoding 20,000- 30,000

cells. Throughout the hydrogel bead prep, if you feel that you are potentially removing

beads with the supernatant, save your supernatants and spin them at 1000g at the end

of your experiment to recover the beads. For example, wrap a 15ml tube in aluminum foil

and dispense all supernatants into the tube. At the end of the washing, spin down the

tube and see if there are any BHMs to be recovered. Add equal volume of Storage Buffer

to the recovered BHMs. These beads can be treated like the other BHMs and rewashed.

d) Wash the BHMs three times as follows:

I. Add 1 mL of washing buffer.

II. Vortex briefly.

III. Centrifuge at 1,000g for 1 min.

IV. Remove the supernatant, do this carefully so as not to disturb the bead pellet.

V. In the last wash, leave ~500 μL in the tube.

e) Add 500 µL of 2x Gel Concentration Buffer, vortex for ~6 seconds, and spin the BHM

suspension at 5,000g for 1 min.

f) Using a p1000 pipette remove as much of the supernatant as possible. 250 μL of close-

packed BHMs should now concentrate to around 150μL.

Critical: Minimize the exposure of BHMs to light prior to encapsulation. Ideally all

handling will take place in dim lighting. Tubes holding the BHMs should be kept away

from direct light and covered, e.g., wrapped in foil.

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g) Spin down the tube at 5,000g for 1 min, and use a p200 pipette tip to remove any remaining supernatant. Note – When a sufficient amount of supernatant is removed, there should be no visible liquid when the tube is tilted.

h) When finished with hydrogel bead prep, put the remaining BHMs, covered from light, at

4C. Do not leave BHMs sitting on bench top in normal lab lighting Note: Unused BHMs can be recovered at end of experiment. Loading excess BHMs is recommended to ensure a proper amount of BHMs for the experiment.

3. Cell and RT/Lysis Buffer Preparation

a) Adjust the concentration of cells to be 100,000 cells/mL, or less, in 1X PBS containing density matching reagent (Optiprep). The cell suspension should have an 18% optiprep to cell suspension. Note – As much as possible, keep the cell sample cool on ice while preparing for a run. When possible, prepare more cell phase than you anticipate you will use, ideally a volume of 200-500 μL. When the number of available cells is very low, e.g., a total number of 1,000 cells, use a final volume of at least 100 μL.

b) Prepare the calculated amount of RT/Lysis mixture. Click on the “?” in the Thor software and the breakdown of the RT/Lysis mixture will be given.

c) On ice, prepare the RT/lysis mix by mixing 1.3x RT premix with MgCl2, DTT, RNaseOUT, and SuperScript III according to the calculated volume. Here, we specify the appropriate volume for 100 μL of RT/Lysis Mix:

Table 1. Components and specified amounts of the RT/Lysis mix for 100 μL

Reagent Amount (μL) Final Concentration 1.3x RT premix MgCl2 (1.0 M)

76 1.1

1x 11 mM

DTT (0.1 M) 6.9 6.9 mM RNaseOUT (40 U/μL) 5.65 2.26 U/μL SuperScript III (200 U/μL) 10.35 20.7 U/μL 100

Critical: Add RNAseOUT and SuperScript III just before encapsulation to reduce the

time on ice. Avoid introducing air bubbles by pipetting gently.

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4. Loading the Microchip

a) Input the desired number of cells to encapsulate and the ratio of beads to cells. The software will compute the volume of beads necessary. Aspirate more BHMs than intended, all extra beads will be recovered at end of experiment. If multiple experiments are intended, input the total number of cells to encapsulate. Note – With small amounts of BHMs, monitor the tubing in the Eppendorf closely to make sure the tip remains fully immersed in the pellet while loading. Aspirate excess beads. If necessary, spin in table top microfuge to re-pellet BHMs.

b) Prime tubing of Beads pump by, pressing “Prime Beads” and wait until a droplet of fluid forms at the tip of the tubing.

c) Let the pump run for 5 seconds and press “Stop” on the software. If there are noticeable air bubbles in the BHM tubing, continue to prime until the air bubbles have been primed out.

d) Inspect the tip of BHM tubing to ensure there are no air bubbles in the tip.

e) Insert the tip of the tubing into the concentrated BHMs and cover beads with a piece of parafilm. Make sure the tubing remains firmly inserted into the tube.

f) Press “Load Beads”. The pump will automatically stop once reached the desired volume.

Recommended: If microchip silanization in needed, this is a good space in the workflow.

g) Handling the special pipette tips cleanly, attach new tips to the luer connectors of the cell and RT Mix syringe. Place the tips inverted in the blue clip on the microscope illumination arm.

h) On the software under the tab “Reagents”, press the button “Load Colored Oil”.

i) If there is still an air gap in the tip, press “Prime Colored Oil” until a drop comes out the top. Press “Prime Colored Oil” again to stop the pump. Wipe the tips clean with a lint free lens wipe.

j) Once your cell and RT/Lysis mixes are prepared, transfer the Eppendorf tubes to the chiller block.

k) Place the chiller block containing the Eppendorf tubes on the right side of the stage.

Critical: Periodically check to make sure the tip of the tubing stays fully immersed in the BHM pellet. If the tubing tip comes out of the BHM pellet, stop the pump, make sure there are no air bubbles in the tip, and re-immerse the tubing tip into the pellet and resume the BHM aspiration.

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l) Unclip the tip filled with colored oil and place them, tips downward, in their appropriate tubes

m) When ready, press “Load Cells” followed by “Load RT Mix”.

n) Once the solutions are loaded, place tips in the blue clip on the right side of the stage. Wipe tips clean with a lens wipe. Press “Next” to access the Prime Tab.

o) Prime the Cell tip by pressing “Prime Cells” until a droplet forms, then press the button again to stop priming. Wipe the tip clean with a low lint wipe. Insert it into the cell inlet.

p) Repeat for previous step for; RT Mix, Beads, Droplet Oil. Refer to “?” on software for graphic of microchip.

q) Place a 20cm long collection tubing in the collection outlet to transfer the emulsion to an Eppendorf tube labeled “Waste” (Figure 7.).

Figure 2: Microfluidic device layout

Caution: If chiller block is in -20C freezer, allow the block to warm up to keep reagents

from freezing.

Figure 3: A fully connected microchip

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5. Initiating Gel Bead Encapsulation

a) Start by clicking “Prime Chip” in the software window (Figure 4). Prime Chip starts the

flow of fluid into the microchip to push out the air bubbles in preparation for starting an

experiment. Table 2 below details the flow rates to use in each phase of device

operation. Note – These flow rates are preset in the Thor software.

Figure 4: Image of Prime Chip tab in Thor software

Table 2: Microdevice Flow Rate Conditions

b) Once all the air is pushed out of the device, the RT/Lysis mixture, Cells and Droplet Oil

will flow through the chip first, followed by the BHMs. Note: It is normal for drops not to

Beads

Oil

Cells

RT mix

Prime Chip (μL/h) 100 200 200 200

Run Experiment (μL/h) 50 500 250 250

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form at this stage. If drops still do not form in the “Run InDrop”

tab, please refer to troubleshooting guide.

c) Once beads are flowing into the bead inlet and closely packed as shown in Figure 5a,

click “Next”. This will adjust the flow rates to preset values that can be overridden and

changed as needed.

Figure 5: BHMs (a) before encapsulation and (b) after encapsulation with cells

6. Optimizing Gel Bead Encapsulation

a) In order to reach BHM encapsulation rates >90% it is important to adjust the flow rate

of the BHMs. It may be necessary to fine tune the flow rate in increments of 5 μL/h. If

the occupancy is below 50%, turn the flow rate up in increments of 10 μL/h. Note – The

occupancy rate is measured by taking a video of the drops downstream from the drop

forming region. Counting the drops and hydrogel beads per drop determines the gel

bead encapsulation percentage as shown in Figure 5b. See troubleshooting guide for

tips on achieving a high occupancy.

b) When the highest occupancy of single beads in drops has been achieved, ensure all

other flows are stable and press “Start”. When the waste timer has reached “0:00”

move the collection tubing to a new 1.5mL Eppendorf tube containing 200uL of mineral

oil. Sample collection should be done in a cold block or ice (Figure 7.)

a b

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c) Continue to monitor the gel bead-cell encapsulation with the high-speed camera or strobe mode. Adjust flow rates, if necessary, to maintain optimal operating conditions. There should be at most 1 BHM in each droplet and about 90% of all droplets should contain beads (>70% bead occupancy should be sufficient for most experiments).

d) Avoid having two BHMs per droplet. Adjust the flow rate of BHMs in 5 μL/h increments to ensure that these conditions are maintained. BHM occupancy can be determined from short movies recorded at the outlet of the microfluidic device or simply monitoring the droplet flow using the Strobing Mode or pausing feature in the software. The Strobing Mode will show the flow conditions at 2 fps.

Figure 6. Layout of Thor software identifying key features

e) Ensure that the droplet size and formation rate remain constant. Confirm the droplet size. Droplet volume in picoliters (pL) is given the calculation below.

Critical: Mineral oil is necessary to prevent droplet coalescence due to evaporation. Also, Keep the emulsion cold by using the chiller block or small ice bath

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f) By using the flow rates indicated in Table 2, the droplet size should be 1.0-3.0 nL and can be adjusted by changing the flow rate of the droplet-making oil, Cells and RT/Lysis mix. Use a timer to determine the proper video length, in seconds.

g) Verification of a low cell doublet rate can be achieved by estimating the number of cells per droplet using the cell flow rate, the cell density, and the droplet volume, or by visual inspection of a sample of droplets using a hemocytometer.

h) Once the desired number of cells is encapsulated, press “End Run”, unplug the tubing

from the outlet. Let the emulsion in the tubing drain into the collection tube by gravity.

i) Disconnect the BHMs and place the tip back in the holder.

j) Disconnect the Cell and RT Mix and place the tips in the blue clips on the right side of the

stage.

k) Disconnect the Droplet Making Oil and place it in its holder.

l) Press “Next” to move to the Shutdown tab.

V = (F x t)/(c x N)

F

T

c

N

V

= Sum of flow rates of all aqueous phases

= Time (sec)

= 3.6 sec µL /(pL)(h)

= droplet count (in t seconds)

= Droplet volume in picoliters

Figure 7. Set up for collecting sample

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m) Place the Cell and RT Mix tips into a waste container and press the button “Dispense Cells”

and “Dispense RT Mix”. When the remaining solutions have been dispensed, press the

buttons again to stop dispensing.

Note: These dispense at a fast flow rate, it will likely only take one or two seconds

to dispense remain solutions.

n) Once the aqueous phases have been dispensed and tips are full of colored oil, place the

tips with, the open end pointed up, on the illumination arm of the microscope and and

press “Withdraw Colored Oil”.

o) If NO other encapsulation is being done, press “Dispense Beads” into a 1.5 mL Eppendorf

tube labeled “Recovered BHMs”. Once ~100ul has been dispensed, press “Dispense

Beads” to stop pump. Add equal volume storage buffer, cover and return beads to 4C.

p) If doing another encapsulation: DO NOT dispense beads in previous step.

q) Once the colored oil has been withdrawn from the Cell and RT mix tips replace the tips

with new ones.

r) If all experiments are complete, remove and dispose of luer lock tips.

s) Go back to tab labeled “Setup” and follow the protocol from the beginning. If beads are

already loaded, press the “Next” button on the beads tab without loading more beads.

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7. cDNA Synthesis Time: 3 hours

To release the photo-cleavable barcoding oligos from the BHMs, keep the collection tube on ice,

expose the collected droplets to UV (6.5 J/cm2 at 365 nm). Using the specified lamp shown in

figure 8, fill a Styrofoam container with ice and place the sample tube horizontally on ice. It is

important that the tube is centered under the lamp Place the bulb 3 to 5 cm above the tube and

irradiate for 10 min, refer to Figure 8.

Figure 8: UV lamp on top of samples to

break the photocleavable linker

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a) If using the 1CellBio UV Device, remove cap, insert 1.5mL Eppendorf into hole on top of

box. Press button, UV light will stop after 10 seconds. See Figure 9

CAUTION: DO NOT LOOK DIRECTLY INTO UV LIGHT. DO NOT TURN ON UV LIGHT

WITHOUT CAP OR 1.5ML TUBE INSERTED.

Figure 9: Removal of the cap (A), place 1.5mL inside hole (B), turn on UV light (C).

b) For efficient cDNA synthesis, incubate the tube for 1 hours at 50°C.

c) Terminate the reaction by heating for 15 min at 70°C.

d) Remove from heat, and remove as much of the mineral oil and residual droplet-making

oil as possible with a pipette (Figure 10).

Figure 10: All layers can be seen (a), after removal of oil (b) and after breaking emulsion (c)

e) If necessary, divide the emulsion into fractions containing the desired number of cells.

For example, if 4000 cells were barcoded, divide the entire volume of the emulsion in

A B C

MineralOil

EmulsionDropletOil

Emulsion

DropletOil

a b c

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two equal parts to get 2000-cell libraries. Using conditions described in this protocol, 35

μL of emulsion typically contain ~1000 barcoded cells.

f) Break the emulsion by adding equal volume of demulsifying agent as the sample

collected. Vortex for 5 seconds and centrifuge at 1000g for 1 min. This step can be

repeated if emulsion is difficult to break. The tube should look like Figure 9c.

g) The cDNA contained in the aqueous phase is now ready to undergo library preparation.

h) Remove as much of the bottom layer (the oil layer) as possible without aspirating any of

the aqueous layer.

i) Following manufacturer’s instructions, clean each single cell library aliquot using a Qiagen

MinElute Reaction Cleanup column. Elute each sample in 12 µL of Qiagen buffer EB or in sterile

water. [Note – If the encapsulation was less than 4 minutes add 20 µL of sterile water to the

sample to bring the volume to the appropriate amount and proceed to Qiagen clean up]

j) At this point, the tubes can be stored at -80°C for at least 3 months, or sequencing

libraries can be prepared from the samples immediately.

8. Instrument Shutdown

a) Flush the black BHM tubing by placing the tip in a collection tube and pressing “Dispense beads” in Shutdown tab. The tube can be treated as a recovery tube for the BHMs. Once the flush is complete, place the tubing back into the holder.

b) Add equal volume of Storage Buffer to the tube containing the recovered beads.

c) Unplug the oil tubing and place the tube in one of the tubing holders on the left side of the microscope stage.

d) If any Cell or RT Mix solutions are left over, evacuate the tips by pressing “Dispense Cells and “Dispense RT Mix”.

e) Once all the aqueous solutions are dispensed, hang the tips, open end up, in the blue clips on the microscope arm, and press “Withdraw Colored Oil”.

f) Once all colored oil is out of the tips, remove and discard the used pipette tips.

g) Insert the end caps into each tubing and place the tubing in one of the tubing holders on the microscope. Note – The syringe assemblies should not be left on the instrument for more than two weeks.

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Notes and Troubleshooting Guide No fluid is coming from one of the channels

-Due to the viscosity of the BHMs, they typically are the last to show up. -Check connection to the microchip -Check for air bubbles in the tip -Increase the flow rate of that channel by 300ul/hr -Stop all pumps, detach tip from microchip and re-prime using the “Prime” tab.

Ensure there is fluid flowing from the tip. Drops are not forming in “Run InDrop” mode

- Ensure there is a tight seal between the Droplet Making Oil tip and the PDMS.

- Make sure there is a sufficient amount of Droplet Making Oil in the syringe. Refill if necessary

- Increase Droplet Making Oil flow rate by 300ul/hr. When drops begin to form, lower to recommended flowrate

Beads are not packing

- Sometimes the beads do not come into the chamber and pack immediately.

- Increase the bead flowrate by 50ul/hr - Supernatant may have been left on the top of the pellet during

preparation - The overlapping structure will start at the top of the chamber and move

towards the channel until the entire chamber is filled with fully packed beads.

- If more than 2 mins is required to achieve a sufficient bead pack, turn down cell and RT/Lysis mix flowrates until beads are sufficiently packed. Turn Cell and RT/Lysis flowrates back when optimizing for single bead occupancy

Single bead occupancy dropped dramatically during encapsulation - Beads may have become unpacked. Increase bead flowrate by 50ul/hr.

Beads should start to become more overlapped in chamber. Do not collect the emulsions while changing bead flowrate. Change tubing to the “Waste” tube and wait until beads have become sufficiently packed. Once packed, lower bead flowrate in increments of 10ul/hr

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until optimal bead occupancy is reached and continue to collect emulsion.

Tips for achieving a high percentage of drops containing a single bead.

- Viewing the beads in strobe mode allows viewing of beads in droplets in real time.

- To visualize the drops better, adjust the brightness by using the small knob on the left side of the microscope

- In strobe mode, pressing the pause button freezes the frame and allows visualization of the drops captured in that particular frame. Counting the single beads in drops is a good way to get an idea of the occupancy percentage. Repeat multiple times to get a bigger sample of the population of drops.

- The videos can be played back in Thor, VLC Media Player and ImageJ. - To play a video back in ImageJ, drag the video from “My Documents” to

the ImageJ utility bar. - Taking a video in high speed mode and playing back and lowering the

frames per second (FPS) allows the visualization of each individual drop.

Tips to avoid wetting

- Wetting occurs due to an incomplete or insufficient surface modification.

- Silanizing the chip immediately before encapsulation will decrease chances of wetting

- The symptoms of wetting include: a. Droplet polydispersity b. Small satellite droplets around larger droplets c. Jetting of the aqueous phase d. Tails or sharp looking edges to the droplets

- Turning up the Droplet Making Oil by 50ul/hr - If flowing Cells and RT Mix above 200ul/hr, reduce to 200ul/hr and

Droplet Oil to 400ul/hr. - A device should be aborted if all of the above and attempted and the

wetting is causing BHM occupancy to fall below 70%. - If device is being aborted, disconnect each channel in the same way

they were connected (i.e. Cell, RT, BHM, Oil) - Re-prime all the reagents before connecting to the next device.