Drop-Chip Molecular Biology

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Preparation of unlabeled chromatin

  • Suspend 100M K562 cells in 1mL of 1x digestion buffer.
  • Lyse @ 4C for 10min
  • MNase @ 37C for 15min
  • Inactivate by adding 40uL of 0.5M EGTA
  • Centrifuge for 5min at max speed and separate the chromatin supernatant
  • Mix 1mL of chromatin with 1mL of 2x stopping buffer.

 

ChIP

Separating the emulsion

  • Starting material – 20uL oil, 20uL drops, 50uL stopping buffer, 25uL unlabeled chromatin
  • Add 10uL demulsifier (1H,1H,2H,2H-perfluoro-1-octanol)
  • Centrifuge @1000 rpm for 30sec and transfer the top aqueous phase to another well

 

Immuno-Precipitation

  • Starting material – 20uL of magnetic beads conjugated to 1-3uL of antibody
  • Add to 100uL of chromatin from 1)
  • Incubate @4C overnight
  • Wash twice with 100uL of low-salt immune complex
  • Wash twice with 100uL of high-salt immune complex
  • Wash once with 100uL of LiCl immune complex
  • Wash once with 100uL of TE (10mM Tris-HCl)
  • Wash once with 21.5uL of TE (10mM Tris-HCl)

 

​Amplification

  • Starting material – precipitated chromatin in 21.5uL of TE

 

PacI digestion

  • Add 1uL of PacI restriction enzyme and 2.5ul of NEB Buffer 1
  • 37C for 2hrs, @ 65C for 20min

 

Chromatin elution

  • Add 25uL of 2X elution buffer and 3uL of Rnase
  • 37C for 20min
  • Add 3uL of Proteinase K
  • 37C for 2hrs, 65C for 30min

 

DNA purification using 1.5X AMPure XP beads 

 

Single-Cell PCR 

  • 5uL Hercules buffer 5x
  • 1.5uL SC-PCR primers 50uM each
  • 1uL DMSO 
  • 0.5uL 25mM DNTPs
  • 0.5uL Hercules Taq Enzyme
  • 16.5 purified DNA
  • Thermocycling:
    • hot start - 95C 3min
    • denature - 95C 30sec ]
    • anealing - 55C 10sec   } X 14 cycles
    • elongate - 72C 1min   ]
    • final extension - 72C 10min

 

DNA purification using 1.1X AMPure XP beads 

 

dephosphorylate 5' end

  • 1uL Antarctic Phosphatase enzyme
  • 2.5uL Antarctic Phosphatase Buffer
  • 21.5uL purified DNA
  • 37C 30min 

 

DNA purification using 1.1X AMPure XP beads 

 

BciVi digestion

  • 1uL BciVi enzyme
  • 2.5uL of NEB Buffer 4
  • 21.5uL purified DNA 
  • 37C 1hr

 

DNA purification using 1.1X AMPure XP beads 

  • reduce sample volume to 4uL via evaporation

 

Ligation of Illumina adapters

  • 0.5uL Quick Ligase
  • 6uL of 2X Quick Ligation Reaction Buffer
  • 1.5uL Illumina adapters diluted 1:150
  • 4uL purified DNA 
  • RT 15min

 

DNA purification using 0.7X AMPure XP beads 

 

PacI digestion

  • 1uL PacI restriction enzyme 
  • 2.5ul NEB Buffer 1
  • 21.5uL purified DNA 
  • 37C 2hrs
  • 65C 20min

 

DNA purification using 0.7X AMPure XP beads 

 

Illumina PCR 

  • 12.5uL PfuUltra II Hotstart PCR Master Mix
  • 0.5uL of Illumina Primers at 25uM 
  • 12 purified DNA
    • Thermocycling:
    • hot start - 95C 3min
    • denature - 95C 30sec ]
    • anealing - 55C 30sec   } X 14 cycles
    • elongate - 72C 1min   ]
    • final extension - 72C 10min

 

DNA purification using 0.7X AMPure XP beads

 

Sequencing

  • illumina Hiseq 2x60 bp paired end reads.
  • The first 11 sequencing cycles are dark.

2X labeling buffer (200uL)

concentration
reagent stock final volume comment
reagent stock final volume comment
PEG 400 100% 30% 60 uL
Quick Ligase buffer (NEB, USA) 10x 2x 45 uL
ATP 10mM 1.75mM 35 uL
EndIt enzyme (Epicentre USA) 16 uL
Fast Ligase (Epicentre, USA) 16 uL
EGTA 0.5M 40mM 16 uL
dNTPs 10mM 0.5mM 10 uL
sodium butyrate 1M 10mM 2 uL

2X digestion buffer (100mL)

concentration
reagent stock final volume comment
Triton® X-100 10% 2% 20 mL
Tris-HCl 1M, pH 7.5 100mM 10 mL
NaCl 5M 300mM 6 mL
sodium deoxycholate 5% 0.20% 4 mL
CaCl2 1M 10mM 1 mL
H2O 59 mL
sodium butyrate 1M 10mM 25uL in 2.5mL of 2X buffer
Micrococcal Nuclease 100% 0.04% 1uL in 2.5mL of 2X buffer

carrier buffer (1mL)

concentration
reagent stock final volume comment
digestion buffer 2x 0.5x 250 mL
PEG 400 100% 18% 175 mL
EGTA 0.5M 20mM 40 mL
PBS 1x 0.54x 535 mL

stopping buffer (100mL)

concentration
reagent stock final volume comment
Triton® X-100 10% 1% 10 mL
EGTA 0.5M 30mM 6 mL
EDTA 0.5M 30mM 6 mL
Tris-HCl 1M, pH 7.5 50mM 5 mL
NaCl 5M 150mM 3 mL
sodium deoxycholate 5% 0.10% 2 mL
H2O 68 mL

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