Micro-C XL Protocol
I. First Crosslinking
- Culture 100 mL of yeast to the midlog stage, OD=0.55 o/n.
- Add 37% formaldehyde directly to the culture to 3% of a final concentration.
- Shake the culture at 210 rpm for 15 min at 30˚C (FA only) or 10 min at 30˚C (Dual crosslinking).
- Quench the crosslinking by adding 10 mL of 2.5M Glycine.
- Incubate for 5 min at room temperature.
- Centrifuge the cells at 4000 rpm for 5 min at 4˚C.
- Pour off the medium and wash the cells in 50 mL of sterile water by vortexing.
- Centrifuge the cells at 4000 rpm for 5 min at 4˚C.
- Pour off the water.
II. Permeabilize the cell wall
- Resuspend the cell pellet in 10 mL of Buffer Z and add 7 µL of 2-Mercaptoethanol (final 10mM).
- Add 250 µL Zymolyase solution (final 250 µg/mL).
- Shake the tube at 210 rpm for 40 min at 30˚C.
- Centrifuge the cells at 4000 rpm for 10 min at 4˚C.
- Aspirate the supernatant with a vacuum suction.
- Rinse the permeabilized cells by 5mL cold 1× PBS.
- Centrifuge the cells at 4000 rpm for 2 min at 4˚C.
- Aspirate the supernatant with a vacuum suction.
III. Second Crosslinking
- Freshly prepare the long crosslinker stock and working solution as below:
Crosslinkers MW Spacer (Å) Stock Working
DSG 326.26 7.7 0.3M in DMSO 3mM in PBS
EGS 456.36 16.1 0.3M in DMSO 3mM in PBS
- Resuspend the cells homogenously by 5 mL of working solution.
- Rotate the tube for 40 min at 30˚C.
- Quench the crosslinking by adding 1 mL of 2.5M Glycine.
- Centrifuge the cells at 4000 rpm for 10 min at 4˚C.
- Aspirate the supernatant with a vacuum suction.
- Rinse the permeabilized cells by 5 mL cold 1× PBS.
- Centrifuge the cells at 4000 rpm for 2 min at 4˚C.
- Aspirate the supernatant with a vacuum suction.
- The crosslinked pellet can be store at -80˚C for few months.
IV. Chromatin fragmentation
- Resuspend the cell pellet in 200 µL of MBuffer#1 (freshly complete).
- Add the appropriate amount of MNase to digest the chromatin to > 95% mononucleosomes.
- Incubate the tube for 20 min at 37˚C.
- Add 2 mM EGTA and incubate the tube for 10 min at 65˚C to stop the MNase activity.
- Here, you can continue processing the sample in multiple ways prior to Micro-C sequencing library generation, depending on the desired experimental design. Micro-C has been successfully carried out using three chromatin preps: 1) Total chromatin. 2) Supernatant. 3) Pellet. Any of these fractions can be subjected to the following Micro-C protocol, although we note that optimal signal-to-noise is achieved using relatively insoluble (Pellet) chromatin.
V. Chromatin cleaning and concentration
- Total chromatin:
- Transfer the whole sample into the 0.5 mL Amicon 10K spin column.
- Concentrate the sample at 16000xg for at 4˚C until the volume goes down to ~ 50 µL.
- Wash / pipette the sample by 450 µL MBuffer#2.
- Repeat wash step 2 - 3.
- Concentrate the sample at 16000xg for at 4˚C until the volume goes down to ~ 30 µL.
- Add BSA to 1× final concentration.
- Supernatant:
- Centrifuge the tube at 16000xg for 5 min at 4˚C.
- Collect the supernatant.
- Concentrate the sample by the 0.5 mL Amicon 10K spin column at 16000xg for at 4˚C until the volume goes down to ~ 50 µL.
- Wash / pipette the sample by 450 µL MBuffer#2.
- Repeat wash step 2 - 3.
- Concentrate the sample at 16000xg for at 4˚C until the volume goes down to ~ 30 µL.
- Add BSA to 1× final concentration.
- Pellet:
- Centrifuge the tube at 16000xg for 5 min at 4˚C.
- Collect the pellet.
- Resuspend the pellet in 1 mL MBuffer#2.
- Centrifuge the tube at 16000xg for 5 min at 4˚C.
- Aspirate the buffer with a vacuum suction.
- Repeat wash steps 3 - 5.
- Resuspend the pellet to 30 µL of MBuffer#2 + final 1× BSA (or NEBuffer 2.1).
VI. Repair and label the end of chromatin fragments
- De-phosphorylation
Total 32.5µL Final condition
Chromatin sample 30µL 50mM NaCl, 10mM Tris, 10mM MgCl2, 1X BSA
1U/µL r-Shrimp alkaline phosphatase 2.5 2.5U
Incubate for 45min at 37°C.
Inactivate for 5min at 65°C.
- End-Chewing
Total 42.7µL Final condition
Chromatin sample from VI.1 32.5µL -
10X NEBuffer#2 3 70mM NaCl, 14mM Tris, 14mM MgCl2
100mM ATP 0.5 1mM ATP
200X BSA 0.2 1X
0.1M DTT 1 3mM
3U/µL T4 DNA polymerase 2.5 7.5U
10U/µL T4 PNK 3 30U
Incubate at 37°C for 7min.
- End-labeling
Total 100µL Final condition
Chromatin sample from VI.2 42.7µL -
0.4mM Biotin-dATP 25 100µM
0.4mM Biotin-dCTP 25 100µM
10mM dTTP + dGTP 1 100µM
10X T4 DNA ligase buffer 6 30mM NaCl, 35mM Tris, 12mM MgCl2, 7mM DTT
200× BSA 0.3 1× BSA
PCR machine: Incubate for 25min at 25°C 15min at 12°C 4°C.
Add EDTA (final 30mM) and heat inactivation for 20 min at 65°C.
VII. Proximity ligation and Remove unligated ends
- Ligation
Although in our test Micro-C in “pellet” can be scaled down to a 1 mL ligation reaction, we suggest using at least 2.5 mL for routine experiments.
Total 2.5mL
(pellet) 10mL
(Total & Sup) Final condition
Chromatin sample from VI. 100L 100µL -
Water 2122 8809 -
10× T4 DNA ligase buffer w/ ATP 250 1000 1×
200× BSA 12.5 50 1×
1M MgCl2 3 3 Equal to the moles of EDTA from previous part.
400U/µL T4DNA ligase 12.5 38 1.5 – 2U/µL
Incubate for 60min at room temperature.
Pellet: Centrifuge the pellet by 16000xg for 10min at 4°C.
Total/Sup: 15mL Amicon 30k concentrates sample by spin at 4000g for 40min at 4°C.
- Remove the biotin-dNTP at unligated ends
Total 100µL
(pellet) 280µL
(Total & Sup) Final
Chromatin sample - 250 -
10× NEBuffer#1 10 28 1×
Water 89 - -
100U/µL Exonuclease III 1 1 100U
Incubate for 5 min at 37°C.
- Reverse crosslinking
Add 20X proteinase K to 1× final concentration.
Incubate for overnight at 55°C.
VIII. Dinucleosomal DNA purification
- Phenol:Chloroform:Isoamyl Alcohol extraction twice spin at 19800xg for 10min.
- Ethanol precipitation: 0.1x volume of sodium acetate and 2.5× volume of 100% ethanol -80°C for > 1hr spin at 19800xg for 15min at 4°C wash pellet by 75% ethanol spin at 19800xg for 5 min at 4°C Air dry pellet for 10min.
- Resuspend pellet in 50L of TE buffer (+ 1× RNase solution) and incubate for 30min at 37°C.
- ZymoClean to purify DNA.
- Run DNA samples on 3% Nusieve agarose DNA gel.
- Size selection of the band between 250 – 350 bp.
- ZymoGel purification and dissolve final product in 17µL of elution buffer.
- Quantify the input DNA by Qubit.
IX. Library construction by “with-bead” method
- End-it
Total 25µL
DNA 17
10× End-it buffer 2.5
10× ATP 2.5
10× dNTP 2.5
End-it enzyme mix 0.5
Incubate for 45 min at room temperature.
2× Ampure XP purification.
- A-tailing
Total 25µL
DNA 16
10X Exo- Klenow buffer 2.5
1mM dATP 5
Exo- Klenow fragment enzyme 1.5
Incubate for 30min at 37°C.
2× Ampure XP purification. (PEG/NaCl solution: 20% PEG, 2.5M NaCl)
- Adapter ligation
Total 15µL
In-line / Indexing Adapter Ratio of Adapter:Input DNA = 10:1 – 50:1
Water to total 15µL
10× Fast-link DNA ligase buffer 1.5
10× ATP 1.5
Fast-link DNA ligase 1
Incubate for > 2 hr at room temperature.
Add 10µL of EB to total volume 25 µL.
2× Ampure XP purification and elute DNA in 150 µL of water.
- Streptavidin beads purification
- Wash 2.5 µL of beads per sample (100mL culture) by 1× TBW twice.
- Resuspend the washed beads in 150 µL 2× BW.
- Mix with 150 µL of adapter-ligated DNA sample.
- Rotate for 15 min at room temperature.
- Wash by 500 µL of 1× TBW twice.
- Rinse by 200 µL of MBuffer#2.
- Resuspend in 15 – 20 µL of EB buffer.
- On-beads PCR
Total 10µL
(scalable)
Streptavidin-Biotin-DNA sample 1
Water 3.5
2× KAPA HiFi Hot Start Mix 5
10µM PE1 primer 0.25
10µM PE2 primer 0.25
Denaturation 98°C 45sec
8-12 cycles 98°C 15sec
60°C 30sec
72°C 30sec
Extension 72°C 1min
4°C Hold
Check the size and quantity of library by DNA gel or Fragment analyzer.
Size-selection of dimer size library by 3% Nusieve agarose DNA gel or 1:1 Ampure XP beads purification.
X. Deep sequencing by Illumina PE-50