DropSynth bead barcoding protocol (version 2.0 Aug 2019)

This protocol can be performed using 1x 384-well plate to generate 384 unique barcoded
beads, or 4x 384-well plates to generate 1536 unique barcoded beads. Though the process canbe done by hand, it is helpful to use a Rainin Liquidator 96 for liquid handling steps.
Reagents Required (384-plex):
1. 240 ​µL​ 100 ​µM​ anchor oligo (​Integrated DNA Technologies)
2. 240 ​µL​ 100 ​µM​ ligation oligo (​Integrated DNA Technologies)
3. 1,056 ​µL​ 10X T4 Ligase Buffer (New England Biolabs)
4. 1 ​µL​ of each 100 ​µM​ barcoded oligo (​Integrated DNA Technologies)
5. 24 ​µL​ T4 Ligase (New England Biolabs)
6. 240 ​µL​ T4 PNK (New England Biolabs)
7. 1500 ​µL​ Streptavidin M270 Dynabeads (Invitrogen)
8. >10 mL ​UltraPure Distilled Water (Invitrogen)
9. >10 mL 2X B&W Buffer
Reagents Required (1536-plex):
1. 960 ​µL​ 100 ​µM​ anchor oligo (​Integrated DNA Technologies)
2. 960 ​µL​ 100 ​µM​ ligation oligo (​Integrated DNA Technologies)
3. 4,224 ​µL​ 10X T4 Ligase Buffer (New England Biolabs)
4. 1 ​µL​ of each 100 ​µM​ barcoded oligo (​Integrated DNA Technologies)
5. 96 ​µL​ T4 Ligase (New England Biolabs)
6. 960 ​µL​ T4 PNK (New England Biolabs)
7. 6,000 ​µL​ Streptavidin M270 Dynabeads (Invitrogen)
8. >40 mL ​UltraPure Distilled Water (Invitrogen)
9. >40 mL 2X B&W Buffer
Prepare 40mL 2X B&W buffer (2M NaCl, 1mM EDTA, 10mM Tris):● 4.675g NaCl salt
● 400 µL UltraPure 1M Tris, pH 7.5 (Invitrogen)● 80 µL UltraPure 0.5 M EDTA, pH 8.0 (Invitrogen)● UltraPure Distilled Water (Invitrogen) to 40 mL
1. Hybridize the anchor, ligation and barcoded oligos:○ Add to the first row of 96-well deep well plate:■ 20 ​µL​ 100 ​µM​ anchor oligo■ 20 ​µL​ 100 ​µM​ ligation oligo■ 80 ​µL​ 10X T4 Ligase Buffer■ 640 ​µL​ ​UltraPure Distilled Water○ Using a Rainin P200 12-channel pipette, add 95 ​µL​ of master mix to all rows ofmaster 96-well plate.
○ Using a Rainin Liquidator 96, distribute 19 ​µL​ of master mix from master 96-wellplate to all wells of a new 384-well plate. The 384-well plate can be adjusted to 4
corners using a Rainin Plate Adapter 384, allowing all wells to be filled from the96-well master plate.
○ Using a Rainin Liquidator 96, transfer 1 ​µL​ from every well of the 100 uM
barcoded oligo plate to every well of the 384-well plate.

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Anneal the mixed oligos on each plate using the following conditions:■ 3 min at 70ºC
■ Ramp down to 60ºC for 1 min, 0.1ºC/sec■ Ramp down to 50ºC for 1 min, 0.1ºC/sec■ Ramp down to 40ºC for 1 min, 0.1ºC/sec■ Ramp down to 30ºC for 1 min, 0.1ºC/sec■ Put plate on ice
Ligate the barcoded oligo to the ligation oligo:○ Add to the first row of a 96-well plate:■ 2 ​µL​ T4 Ligase■ 8 ​µL​ 10X T4 Ligase Buffer■ 70 ​µL​ ​UltraPure Distilled Water○ Using a Rainin P20 12-channel pipette, add 10 ​µL​ of master mix to all rows of amaster 96-well plate.
○ Using a Rainin Liquidator 96, distribute 2 ​µL​ master mix from the master 96-wellplate to all wells of the 384-well plate.
○ Incubate plate at 16ºC for 1 hr or longer, followed by 65ºC for 20 min to heat
inactivate the ligase.
Phosphorylate the barcoded oligo:○ Add to first row of 96-well plate:■ 20 ​µL​ T4 PNK■ 60 ​µL​ ​UltraPure Distilled Water○ Using a Rainin P20 12-channel pipette, add 10 ​µL​ of master mix to all rows of amaster 96-well plate.
○ Using a Rainin Liquidator 96, distribute 2 ​µL​ master mix from the master 96-wellplate to all wells of the 384-well plate.
○ Incubate the plate at 37ºC for 40 min (or longer), followed by 65ºC for 20 min toheat inactivate the PNK.
Bind to beads:○ Prepare 1500 ​µL​ stock Dynabeads M270 Streptavidin, washed, andresuspended in 3000 ​µL​ 2X B&W buffer.
○ Add 200 ​µL​ to first row of 96-well plate.
○ Using a Rainin P200 12-channel pipette, add 25 ​µL​ of master mix to all rows of amaster 96-well plate.
○ Using a Rainin Liquidator 96, add 5 ​µL​ resuspended beads to each well of the384-well plate.
○ Mix overnight with shaking (>2000 RPM) at room temperature.
Pool beads:○ Using a Rainin Liquidator 96, wash each well with 20 ​µL​ 2X B&W buffer 8 times.
○ Using a Rainin Liquidator 96, resuspend each well in 5 ​µL​ 2X B&W buffer.
○ Mix 5 ​µL​ of each well together, making a 1920 ​µL​ mixed barcoded bead pool foreach plate. ​Store these at 4ºC when not in use.