TITLE: DNA Barcoded Hydrogel Bead Synthesis
AUTHORS: Chris Cheshire

[STEPS]
?. [10% (vol/vol) Triton X-100]
Prepare 10% (vol/vol) Triton X-100Combine   of NFW with   of  Triton X-100Store at   for at least 6 months
90 mL
10 mL
0 Room temperature
?. [10% (vol/vol) Tween-20]
Prepare 10% (vol/vol) Tween-20Combine  of NFW with  of Tween-20Store at  for at least 6 months
90 mL
10 mL
0 Room temperature
?. [NaCl]
Prepare 5 M NaClDissolve   of NaCl in  of NFWAdjust final volume to Store at   for at least 1 year
291.65 g
[approx.]
1 L
0 Room temperature
?. [KCl]
Prepare 1 M KClDissolve   of NaCl in  of NFWAdjust final volume to Store at   for at least 1 year
74.55 g
[approx.]
1 L
0 Room temperature
?. [Perfluorooctanol]
Prepare 20% (vol/vol) Perfluorooctanol in HFE-7500 oil (20% (vol/vol)) - 20% PFOCombine   of HFE-7500 oil with   of PerfluorooctanolStore at   for at least 1 year
Perform this step in a fume-hood with appropriate personal protective equipment and avoid contact with skin and eyes when handling this reagent.Store the resulting mixture in a fume cupboard.
8 mL
2 mL
0 Room temperature
?. [DNA elution buffer]
Prepare  of DNA elution buffer  ABCD1ReagentStarting conc.Reaction conc.Volume to add2Tris-HCl1 M10 mM500 μl3EDTA0.5 M100 μM10 μl4NFW49.5 mL5Total50 mLMake into   aliquots and store them at   for up to 1 year.
[approx.]
ABCD1ReagentStarting conc.Reaction conc.Volume to add2Tris-HCl1 M10 mM500 μl3EDTA0.5 M100 μM10 μl4NFW49.5 mL5Total50 mL
1 mL
-20 °C
?. [Tris–EDTA–Tween (TET) buffer]
Prepare  of TET buffer  ABCD1ReagentStarting conc.Reaction conc.Volume to add2Tris-HCl1 M10 mM5 mL3EDTA0.5 M10 mM10 mL4Tween-2010%0.1 %5 mL5NFW480 mL6Total500 mLFilter solution through a  membrane.Store at   for up to 1 year
500 mL
ABCD1ReagentStarting conc.Reaction conc.Volume to add2Tris-HCl1 M10 mM5 mL3EDTA0.5 M10 mM10 mL4Tween-2010%0.1 %5 mL5NFW480 mL6Total500 mL
0 Room temperature
?. [Tris-buffered saline–EDTA–Triton (TBSET) buffer]
Prepare  of TBSET buffer  ABCD1ReagentStarting conc.Reaction conc.Volume to add2Tris-HCl1 M10 mM10 mL3EDTA0.5 M10 mM20 mL4NaCl5 M137 mM27.4 mL5KCl1 M2.7 mM2.7 mL6Triton X-10010 %0.1 %10 mL7NFW822 mL8Total1 LFilter solution through a  membrane.Store at   at least 6 months
1 L
ABCD1ReagentStarting conc.Reaction conc.Volume to add2Tris-HCl1 M10 mM10 mL3EDTA0.5 M10 mM20 mL4NaCl5 M137 mM27.4 mL5KCl1 M2.7 mM2.7 mL6Triton X-10010 %0.1 %10 mL7NFW822 mL8Total1 L
0 Room temperature
?. [1% (vol/vol) Span-80 in hexane]
Prepare  of 1% (vol/vol) Span-80 in hexaneCombine  of hexane with  of Span-80Store at   in a fume cupboard for at least 6 months
100 mL
Perform this step in a fume-hood with appropriate personal protective equipment and avoid contact with skin when handling this reagent.Store the resulting mixture in a fume cupboard.
99 mL
1 mL
0 Room temperature
?. [Hydrogel bead wash buffer]
Prepare  of HB wash buffer  ABCD1ReagentStarting conc.Reaction conc.Volume to add2Tris-HCl1 M10 mM10 mL3EDTA0.5 M100 μM200 μl4Tween-2010 %0.05 %5 mL5NFW980 mL6Total~ 1 LFilter solution through a  membrane.Store at   at least 6 months
1 L
ABCD1ReagentStarting conc.Reaction conc.Volume to add2Tris-HCl1 M10 mM10 mL3EDTA0.5 M100 μM200 μl4Tween-2010 %0.05 %5 mL5NFW980 mL6Total~ 1 L
0 Room temperature
?. [STOP-25 buffer]
Prepare  of STOP-25 buffers  ABCD1ReagentStarting conc.Reaction conc.Volume to add2Tris-HCl1 M10 mM10 mL3EDTA0.5 M25 mM50 mL4Tween-2010 %0.1 %10 mL5KCl2 M100 mM50 mL6NFW880 mL7Total1 LFilter solution through a  membrane.Store at   at least 6 months
1 L
ABCD1ReagentStarting conc.Reaction conc.Volume to add2Tris-HCl1 M10 mM10 mL3EDTA0.5 M25 mM50 mL4Tween-2010 %0.1 %10 mL5KCl2 M100 mM50 mL6NFW880 mL7Total1 L
0 Room temperature
Prechill to   before use.
?. [STOP-10 buffer]
Prepare  of STOP-10 buffer  ABCD1ReagentStarting conc.Reaction conc.Volume to add2Tris-HCl1 M10 mM10 mL3EDTA0.5 M10 mM20 mL4Tween-2010 %0.1 %10 mL5KCl2 M100 mM50 mL6NFW910 mL7Total1 LFilter solution through a  membrane.Store at   at least 6 months
1 L
ABCD1ReagentStarting conc.Reaction conc.Volume to add2Tris-HCl1 M10 mM10 mL3EDTA0.5 M10 mM20 mL4Tween-2010 %0.1 %10 mL5KCl2 M100 mM50 mL6NFW910 mL7Total1 L
0 Room temperature
?. [Hybridization buffer]
Prepare  of Hybridization buffer  ABCD1ReagentStarting conc.Reaction conc.Volume to add2Tris-HCl1 M10 mM5 mL3EDTA0.5 M100 μM100 μl4Tween-2010 %0.1 %5 mL5KCl2 M330 mM82.5 mL6NFW407 mL7Total500 mLFilter solution through a  membrane.Store at   at least 6 months
500 mL
ABCD1ReagentStarting conc.Reaction conc.Volume to add2Tris-HCl1 M10 mM5 mL3EDTA0.5 M100 μM100 μl4Tween-2010 %0.1 %5 mL5KCl2 M330 mM82.5 mL6NFW407 mL7Total500 mL
0 Room temperature
?. [Neutralization buffer]
Prepare  of Neutralization buffer  ABCD1ReagentStarting conc.Reaction conc.Volume to add2Tris-HCl1 M100 mM50 mL3EDTA0.5 M10 mM10 mL4Tween-2010 %0.1 %5 mL5NaCl1 M100 mM50 mL6NFW385 mL7Total500 mLFilter solution through a  membrane.Store at   at least 6 months
500 mL
ABCD1ReagentStarting conc.Reaction conc.Volume to add2Tris-HCl1 M100 mM50 mL3EDTA0.5 M10 mM10 mL4Tween-2010 %0.1 %5 mL5NaCl1 M100 mM50 mL6NFW385 mL7Total500 mL
0 Room temperature
?. [10% (wt/vol) ammonium persulfate (APS)]
Prepare  of 10% (wt/vol) ammonium persulfate (APS)Combine  of ammonium persulfate with  of NFWAdjust final volume to Filter solution through a  membraneAliquots of  can be stored at   for up to 6 months
5 mL
Perform this step in a fume-hood with appropriate personal protective equipment and avoid contact with skin when handling this reagent.
500 mg
4.5 mL
5 mL
100 µl
-20 °C
?. [4× Acrylamide/bis-acrylamide (AB) solution]
Prepare 4x of  of Acrylamide/bis-acrylamide (AB) solution  ABCD1ReagentStarting conc.Reaction conc.Volume to add2Acrylamide solution40 %-2.58 mL3Acrylamide/bis-acrylamide solution40 %-3.6 mL4NFW3.82 mL5Total10 mLFilter solution through a  membrane.Store at   at least 6 months
10 mL
Acrylamide and bis-acrylamide are potent neurotoxins.Perform this step in a fume-hood with appropriate personal protective equipment and avoid contact with skin and eyes when handling this reagent.
ABCD1ReagentStarting conc.Reaction conc.Volume to add2Acrylamide solution40 %-2.58 mL3Acrylamide/bis-acrylamide solution40 %-3.6 mL4NFW3.82 mL5Total10 mL
4 °C
?. [Acrydite-modified primer]
5′-/acrydite/photocleavable spacer/CGATGACGTAATACGACTCACTATAG GGATACCACCATGGCTCTTTCCCTACA CGACGCTCTTC-3′Resuspend and dilute to   in DNA Elution BufferStore at  for at least 1 year
Acrydite-modified DNA primers are light-sensitive, as they contain a photocleavable (1-(2-nitrophenyl)-ethyl-based) moiety. At all times protect the DNA primers from exposure to UV light (Perform in dim illumination and away from UV light sources.
-20 °C
?. [Denaturation solution]
Prepare  of denaturation solution.  ABCD1ReagentStarting conc.Reaction conc.Volume to add2NaOH10 M150 M3.75 mL3Brij-3530 %0.5 %4.2 mL4NFW242 mL5Total250 mLFilter solution through a  membrane.Store at PREPARE FRESH
250 mL
NaOH is highly corrosive; wear appropriate personal protective equipment when handling this reagent.Perform this step in a fume-hood with appropriate personal protective equipment and avoid contact with skin and eyes when handling reagents in this section.
ABCD1ReagentStarting conc.Reaction conc.Volume to add2NaOH10 M150 M3.75 mL3Brij-3530 %0.5 %4.2 mL4NFW242 mL5Total250 mL
0 Room temperature
?. [Carrier Oil (1% surfactant)]
Prepare   of carrier oil mixCombine  of HFE-7500 oil with  of dSurf (2%)Vortex wellStore at  and prepare freshly for the experiment run
2.5 mL
1.25 mL
1.25 mL
0 Room temperature
?. [Carrier oil / TEMED mix]
Prepare  of TEMED mixCombine  of carrier oil with  of TEMEDVortex wellStore at   and prepare freshly for the experiment run
2.5 mL
Perform this step in a fume-hood with appropriate personal protective equipment and avoid contact with skin when handling this reagent.
2.5 mL
10 µl
0 Room temperature
?. [Acrylamide–primer mix]
Prepare  of Acrylamide–primer mix in a  tube  ABCD1ReagentStarting conc.Reaction conc.Volume to add2TBSET buffer??100 μl3AB Solution??250 μl4Acrydite-modified DNA primer250 μM50 μM200 μl510% (wt/vol) APS10 %0.3 %30 μl6NFW420 μl7Total1000 μl
1 mL
2 mL
Acrylamide and bis-acrylamide are potent neurotoxins.Perform this step in a fume-hood with appropriate personal protective equipment and avoid contact with skin and eyes when handling reagents in this list.
Acrydite-modified DNA primers are light-sensitive, as they contain a photocleavable (1-(2-nitrophenyl)-ethyl-based) moiety. At all times protect the DNA primers from exposure to UV light (Perform in dim illumination and away from UV light sources.
ABCD1ReagentStarting conc.Reaction conc.Volume to add2TBSET buffer??100 μl3AB Solution??250 μl4Acrydite-modified DNA primer250 μM50 μM200 μl510% (wt/vol) APS10 %0.3 %30 μl6NFW420 μl7Total1000 μl
?. [Prepare MF Equipment]
Cut tubing for the following lengths using the template guides found in the template bag:2 x lengths of each size of 1/323 x lengths of the 1/16 tube
?. [Prepare MF run]
Place Carrier oil/TEMED mix and Acrylamide–primer mix into p-valve system.Connect the sample tube to p-valve headCover tube with foilSlide green spacer over 1/32 tube to centerThread green space and tube through the cream seal and place the blue plug in reverse over the green spacer facing the cream seal threadPlace 1/32 tubing through the aluminium seal in top until it hits the bottom of tubeSlide the green spacer and seals down until they hit the aluminium threadScrew seal thread until the 1/32 tubing does not move anymorePlace both into tube stand
?. [Acrylamide–primer mix]
Vortex solution to mix and centrifuge -
Centrifuge: 1000 34, 5 s
?. [Acrylamide–primer mix]
Place  of mineral oil on top of the solution
500 µl
?. [Prepare MF run]
Place MF bead device onto microscope mount upside down and secure
?. [Prepare MF run]
Attach flow units and connect conversion adapter to 1/32 tubing coming from each p-valveSlide flow-unit seal over the 1/32 tube coming from p-valve and connect to LHS of flow unit. Tighten until the tube does not move anymoreAttach the short 1/32 tubing to the RHS of the flow unit using the same methodAttach red conversion adapter to the 1/16 tubingAttach 1/32 from RHS flow-unit to the other side of the conversion adaptor
?. [Prepare MF run]
Connecting tubing to MF device including output
?. [Prepare MF run]
Place p-valve head unit over collection tube and place into tube rack. Slide output tubing into top of p-valve but do not seal.Wrap output tube in foil.
?. [Prepare MF run]
Turn on powerTurn on microscope lightingConnect the camera to a laptop and get a live picture
?. [MF run start-up]
TODO
?. [MF run shutdown]
TODO
?. [HB Clean-up]
Remove the top (mineral oil) and bottom (carrier oil) phases from the 2-ml collection tubes, leaving   of polymerized HBs per tube.TODO: PICTURE
[approx.]
?. [HB Clean-up]
Add  of TBSET buffer on top of the creamy-looking emulsion.TODO: PICTURE
500 µl
?. [HB Clean-up]
Release HBs from the emulsion by adding  of 20% PFOVortex well then centrifuge at HBs should appear as a milky (or semitransparent) phase.TODO: PICTURE
1 mL
Centrifuge: 5000 34, 30 s
?. [HB Clean-up]
Remove the bottom 20% PFO phase.Repeat  until the milky phase is transformed into a solid, well-packed, translucent mass on top of the 20% PFO phase.Remove the final bottom 20% PFO phase.
?. [HB Clean-up]
Add  of 1% (vol/vol) Span-80 in hexane Vortex well then centrifuge at This time, the HBs will appear as a solid mass at the bottom of the tube.Remove the hexane layer.Repeat this step.
1 mL
Centrifuge: 5000 34, 30 s
?. [HB Clean-up]
Add  of TBSET buffer and resuspend HBs by vortexing.
1 mL
?. [HB Clean-up]
Transfer HBs to  tubes.
15 mL
?. [HB Clean-up]
Fill tubes with TBSET buffer.Vortex then centrifuge at The HBs will sediment whereas traces of hexane will appear as a milky layer on the top. Carefully aspirate the supernatant, keeping the tip of the serological pipette at the liquid-air interface.
Centrifuge: 3000 34, 3 min
If the HBs do not sediment, extend the centrifuge time.
?. [HB Clean-up]
Repeat  until the top milky layer disappears.
?. [HB Clean-up]
Pass the HBs through a  cell strainer on top of a  tube.Use ice-cold TBSET buffer to facilitate the passage of the beads through the mesh.
50 mL
?. [HB Clean-up]
TODO: QC the bead sizePut 20 microlitre on slide and check in microscope, take picture and use imageJ to check measurement - using python script for size of particles etcget a histogram
?. [Polymerize beads]
Transfer HB tubes to an incubator overnight at  protected from light.
65 °C
?. [HB Clean-up]
Centrifuge at Remove supernatant.Transfer batches of HBs into  tubes and collect remaining into  tube. Wash the sides of the tube with   of TBSET buffer to release  HBs stuck to the side for the  tube.
Centrifuge: 3000 34, 10 min
15 mL
50 mL
1 mL
50 mL
?. [HB Clean-up]
Store at  for up to 6 months protected from light.
4 °C
?. [MF Clean]
TODO
?. [Prepare MF Equipment]
Collect MF equipment:2 x p-valve head block2 x long 1/32 tubing2 x p-valve attachment kitClean heads and o-rings and bring to fumehood
?. [Prepare MF run]
Move tube stand from fume-hood to the MF bench
?. [Prepare MF run]
Adjust lighting, slide position and focus until droplet junction is in ROI and focused/lit properly
?. [Prepare MF run]
Attach blue pressure tubing to p-valves
?. [Bead QC]
Prepare FAM-PE1 probe 5′-/6-FAM/AGATCGGAAGAGCGTCGTGTAGGGAAAGAG-3′ (standard desalting)Prepare a   solution in DNA elution buffer. This solution is stable for at least 1 year at
-20 °C
?. [Bead QC]
Spin down hbsadd    to   tubes and 1.4 mL of QC bufferVortexCent 1 min @ 1,000gremove supermatentadjust final QC buffer vol to Add  of probemix by pipetting up and downincubate room temp for 20 minsWash x 3 - add 1.4ml of QC buffer, vortex, centrifuge 1 min @ 1000g - remove supernatantshould have ~40 left in each tubeSplit into 2 tubesPlace on slide and imageexpose other tube to UV place on slide and image
10 µl
1.5 mL
36 µl
4 µl
?. [QC Buffer]
ABCD1ReagentStarting conc.Reaction conc.Volume to add2Tris-HCl1 M?250 μl3EDTA0.5 M?500 μl4Tween-2010% (vol/vol)?250 μl5KCl1 M1 M49 mL6NFW0 mL7Total50 mL
ABCD1ReagentStarting conc.Reaction conc.Volume to add2Tris-HCl1 M?250 μl3EDTA0.5 M?500 μl4Tween-2010% (vol/vol)?250 μl5KCl1 M1 M49 mL6NFW0 mL7Total50 mL