May 14, 2023
Micro-C protocol for frozen tissue using the Dovetail® Micro-C Kit
- 1Uppsala University
- methods
Protocol Citation: Eric RA Pederson 2023. Micro-C protocol for frozen tissue using the Dovetail® Micro-C Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.14egnz4kyg5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 10, 2021
Last Modified: May 14, 2023
Protocol Integer ID: 47151
Keywords: micro-C, Dovetail, Frozen tissue, use on frozen tissue, frozen tissue, dovetail,
Abstract
micro-C protocol from Dovetail for the use on frozen tissue, in this case
Guidelines
The amount of mononuclease content must be between 30-70%
Materials
Sera-Mag SpeedBeads Carboxylate-Modified Magnetic ParticlesGE HealthcareCatalog #44152105050350
DMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
Pierce DSG No-Weigh Format Thermo ScientificCatalog # A35392
Dovetail Micro-C KitCatalog #21006
Dovetail Filter SetCatalog #BOM-006
Dovetail Hi-C Library Preparation KitCatalog #21004
- 1 ml syringe
DNA Clean & Concentrator™-5Zymo ResearchCatalog #D4003
SPRIselect reagent kitBeckman CoulterCatalog #B23317
16% Formaldehyde Solution (10 ml ampoules)Canemco & MarivacCatalog #0173
Equipment
Cryogrinder set
NAME
OPS Diagnostics
BRAND
CG 08-02
SKU
LINK
Equipment
Bio RS-24 Mini-rotator
NAME
mini-rotator
TYPE
BioSan
BRAND
RS-24
SKU
LINK
Equipment
Thermomixer C
NAME
Eppendorf
BRAND
2231000667
SKU
LINK
Equipment
new equipment
NAME
Qubit 2.0 Fluorometer instrument
BRAND
Q33226
SKU
with Qubit RNA HS Assays
SPECIFICATIONS
Equipment
4200 TapeStation System
NAME
Electrophoresis tool for DNA and RNA sample quality control.
TYPE
TapeStation Instruments
BRAND
G2991AA
SKU
LINK
Protocol materials
Dovetail Filter SetCatalog #BOM-006
Dovetail Hi-C Library Preparation KitCatalog #21004
SPRIselect reagent kitBeckman CoulterCatalog #B23317
Sera-Mag SpeedBeads Carboxylate-Modified Magnetic ParticlesGE HealthcareCatalog #44152105050350
Pierce DSG No-Weigh Format Thermo ScientificCatalog # A35392
DNA Clean & Concentrator™-5Zymo ResearchCatalog #D4003
16% Formaldehyde Solution (10 ml ampoules)Canemco & MarivacCatalog #0173
Dovetail Micro-C KitCatalog #21006
DMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
1X PBS (Phosphate-buffered saline )
EGTACatalog #E8145
Safety warnings
Formaldehyde should be used only under a fume hood, while wearing goggles, gloves and a lab coat.
Be careful not to burn hands with liquid nitrogen. Also should be wearing goggles at that point.
Before start
Complete everything in the pre-epxerimentation section
pre-experimentation
All steps should be performed on ice or at 4 °C .
Autoclave cryogrinder pieces wrapped in foil
pre-cool the cryogrinder pieces
Ensure there is enough liquid nitrogen for the protocol
pre-Step 1
1h 30m
Stage 1: Crosslinking, Digestion and Lysis
As you prepare for Stage 1, keep the following in mind:
Sample preparation takes ~ 1.5 hours.
Before You Begin
The 10X Wash Buffer and 20% SDS might have precipitated in storage.
Incubate these solutions at 37° for 15 minutes or until the precipitate is no longer visible.
Vortex to mix prior to use.
Dilute 10x wash buffer to 1x with ultrapure water.
store at room temperature.
1x wash buffer is stable at room temperature for 2 months.
You need ~ 2 ml of 1x wash buffer per sample for the entire protocol.
Prepare 3 M DSG in DMSO (anhydrous) by dissolving 1 mg of DSG in 10.22 μl DMSO.
DSG is water-insoluble and moisture-sensitive.
Prepare immediately before use.
Do not store DSG in solution.
DMSOMerck MilliporeSigma (Sigma-Aldrich)Catalog #472301
Pierce DSG No-Weigh Format Thermo ScientificCatalog # A35392
Prepare fresh 1x nuclease digest buffer and store at room temperature.
1x nuclease digest buffer is stable for 1 day at room temperature.
You need 50 μl of 1x nuclease digest buffer per sample.
To prepare 1x nuclease digest buffer (50 μl), mix the following components:
| A | B | C | D | E | F | |
| Reagent | 1X | 10% extra | 3X | 5X | 8X | |
| UltraPure water | 40 ul | 44 ul | 132 ul | 220 ul | 352 ul | |
| 10x Nuclease digest buffer | 5 ul | 5.5 ul | 16.5 ul | 27.5 ul | 44 ul | |
| 100 mM MgCl2 | 5 ul | 5.5 ul | 16.5 ul | 27.5 ul | 44 ul | |
| total | 50 ul | 165 ul | 275 ul | 440 ul |
Set the thermal mixer at 22°, shaking at 1,250 rpm.
22 °C
1250 rpm
Thaw 0.5 M EGTA at room temperature.
EGTACatalog #E8145
Vortex to mix prior to use.
Step 1
Weigh out 20 mg of frozen tissue sample.
If using muscle tissue, weigh out 50 mg.
Grind the sample to a fine powder (flour-like consistency) with a mortar and pestle in liquid nitrogen.
Equipment
Cryogrinder set
NAME
OPS Diagnostics
BRAND
CG 08-02
SKU
LINK
Ensure the sample is kept frozen while grinding.
Transfer the ground tissue sample to a 1.5 mL tube containing: 1mL 1XPBS, 10μL0.3 DMSG1X PBS (Phosphate-buffered saline )
Rotate the tube for 10 minutes at room temperature.
Cells should not settle.
Add 62.5 μL 16% formaldehyde.
16% Formaldehyde Solution (10 ml ampoules)Canemco & MarivacCatalog #0173
Rotate the tube for 10 minutes at room temperature.
Cells should not settle.
Spin the tube at 3,000 x g for 5 minutes.
Carefully remove and discard the supernatant. Use caution, the pellet might be loose.
Wash the pellet with a total of 1 mL 1X Wash buffer: first add 200 μL of Wash Buffer and pipette up and down to break up clumps then add the remaining 800 μL.
Pipette up and down to fully resuspend the pellet.
Spin the tube at 3,000 x g for 5 minutes.
Carefully remove and discard the supernatant.
Repeat steps 8 and 9 once, for a total of 2 washes.
After removing the second wash, resuspend the pellet in 1 mL 1X Wash Buffer.
Pipette up and down to fully mix.
Using a 1 mL syringe, gently push the 1 mL resuspended sample through a 200 μm filter into a new 5 mL tube.
Dovetail Filter SetCatalog #BOM-006
If the filter clogs, replace with a new 200 μm filter and continue until all of the sample has been filtered.
Gently pass an additional 1 mL of 1X Wash Buffer though the 200 μm filter into the 5 mL tube.
Your tube should now contain a total volume of ~2 mL.
Using the same syringe but changing to a 50 μm filter, re-filter the 2 mL sample into a new 5 mL tube.
Gently pass an additional 1 mL of 1X Wash Buffer though the 50 μm filter into the 5 mL tube.
Your tube should now contain a total volume of ~3 mL.
Spin the tube at 3,000 x g for 5 minutes.
Carefully remove and discard the supernatant.
Resuspend the pellet in 50 μL 1X Nuclease Digest Buffer
Add 0.5 μL of MNase Enzyme Mix.
Pipette up and down to fully mix.
Incubate the tube at 22° for exactly 15 minutes in an agitating thermal mixer set at 1,250 rpm.
If you are working with a large number of samples, stagger the start of the digestion for each sample by 20 seconds then stop after corresponding 15 minutes
Stop the reaction by adding 5 μL of 0.5 M EGTA.
Pipette up and down to fully mix.
Add 3 μL of 20% SDS to lyse the cells.
Pipette up and down to fully mix.
Incubate at 22° for 5 minutes in an agitating thermal mixer set at 1,250 rpm.
Continue to Stage 2: Lysate QC, page 13.
pre-step 2
Stage 2: Lysate QC
As you prepare for Stage 2, keep the following in mind:
The Lysate QC stage takes ~ 2 hours.
This stage has 2 objectives:
Quantify the lysate to determine the volume of lysate to use in Stage 3.
Confirm that the chromatin was properly digested.
Recommended kit; D5000 HS (Tapestation)
Before You Begin
Verify before use that 100% ethanol was added to the DNA Wash Buffer supplied in the Zymo Research DNA Clean & Concentrator-5 Kit, as directed by the manufacturer.
Program the thermal mixer as follows:
| A | B | |
| Temp | Time | |
| 55 | 15 minutes | |
| 68 | 45 minutes | |
| 25 | Hold |
10X Crosslink Reversal Buffer might have precipitated in storage.
Incubate at 37° for 15 minutes or until the precipitate is no longer visible.
Vortex to mix prior to use.
Step 2
Transfer 2.5 μL of the lysate to a new 1.5 mL tube labelled QC.
NOTE: Store the remainder of the lysate at -80°.
This is the lysate you will be using in Stage 3.
Digest Buffer (50 μL), mix the following components:
| A | B | C | D | E | F | |
| Reagent | 1X | 10% extra | 3X | 5X | 8X | |
| UltraPure water | 45 ul | 49 ul | 147 ul | 245 ul | 392 ul | |
| 10x Crosslink Reversal buffer | 5 ul | 5.5 ul | 16.5 ul | 27.5 ul | 44 ul | |
| Proteinase K | 1.5 ul | 1.65 ul | 4.95 ul | 8.25 ul | 13.2 ul | |
| total | 51.5 ul | 165 ul | 275 ul | 440 ul |
Add to the QC tube 51.5 μL from the master mix of the digest buffer
Pipette up and down to fully mix.
Incubate the QC tube in an agitating thermal mixer set at 1,250 rpm as follows:
| A | B | |
| Temp | Time | |
| 55 | 15 minutes | |
| 68 | 45 minutes | |
| 25 | hold |
(make sure the samples go down to 25° as well)
Pipette up and down to fully mix.
Incubate the QC tube in an agitating thermal mixer set at 1,250 rpm as follows:
Purify the QC sample using Zymo Research DNA Clean and ConcentratorTM-5 Kit (DCC).
Quick spin your QC tube, add 200 μL of DCC DNA Binding Buffer, and mix thoroughly.
DNA Clean & Concentrator™-5Zymo ResearchCatalog #D4003
Transfer the mixture to the Zymo-SpinTM Column placed in a collection tube.
Centrifuge for 30 seconds at 13,000 x g.
Discard the flow-through.
Add to the column 200 μL DCC DNA Wash Buffer
Centrifuge for 1 minute at 13,000 x g.
Discard the flow-through.
Repeat steps 7 and 8 once, for a total of 2 washes.
Transfer the column to a new 1.5 mL tube.
Add 10 μL DCC DNA Elution Buffer directly to the column and incubate for 1 minute at room temperature.
Centrifuge for 1 minute at 13,000 x g. Discard the column.
Your 1.5 mL tube now contains your purified QC DNA.
Quantify the purified QC DNA with a Qubit® Fluorometer and Qubit® dsDNA HS Kit. Based on the Qubit concentration, the total lysate amount (ng) can be calculated as follows:
Total Lysate (ng) = Qubit reading ng/μL x 10 μL (elution volume) x 23.4 (dilution factor)
You will use in Stage 3 a volume of the lysate that corresponds to 1,000 ng. This volume can be calculated as follows:
Volume (μL) = 1000 (ng) x 58.5 (μL) / Total Lysate (ng)
If the total lysate amount is < 1,000 ng, use all of the lysate in Stage 3.
Check the fragment size distribution of your purified QC sample on a TapeStation D5000 HS ScreenTape. Make sure your sample is diluted to 1 ng/μL.
- For optimal nucleosome-level resolution, the digestion profile should contain 40% - 70% mononucleosomes: the first DNA peak, typically in the size range of 50 - 250 bp for TapeStation, should account for 40% - 70% of total DNA (Figure 3). The size range of the peak may vary for other analytical instruments such as Bioanalyzer and Fragment Analyzer. If the digestion profile contains 40% - 70% mononucleosomes, proceed to Stage 3: Proximity Ligation, page 16.
- If the digestion profile contains < 40% mononucleosomes, do not proceed with the rest of the protocol. In this case, re-start the protocol and use 2 μL of MNase Enzyme Mix instead of 0.5 μL in step 14 in Stage 1: Crosslinking, Digestion and Lysis, page 11.
- If the digestion profile contains > 70%, you may proceed to Stage 3: Proximity Ligation, page 16 with caution. The library may include a reduced proportion of long-range information. This profile is likely due to suboptimal cell counting or a significant cell loss in the washing steps after crosslinking (steps 9 to 12 in Stage 1: Crosslinking, Digestion and Lysis, page 11).
pre-step 3
Stage 3: Proximity Ligation
As you prepare for Stage 3, keep the following in mind:
Proximity ligation takes ~ 4.5 hours.
Follow best practices when working with beads
Before You Begin
10X Crosslink Reversal Buffer might have precipitated in storage.
Incubate at 37° for 15 minutes or until the precipitate is no longer visible. Vortex to mix prior to use.
Thaw End Polishing Buffer, 5X Bridge Ligation Buffer, Bridge, and Intra-Aggregate Ligation Buffer at room temperature.
Vortex to mix prior to use.
Prepare fresh 80% ethanol for DNA purification with speed beads for optimal results.
Fresh preparations of 80% ethanol will also be used in Stage 4, DNA Purification, page 24 and Stage 5, Size Selection, page 27.
You need a minimum of 1 mL for all these stages.
Equilibrate TE Buffer pH 8.0, Chromatin Capture Beads, and speed beads to room temperature.
Step 3
3.1 Bind Chromatin to Chromatin Capture Beads
Follow the steps below for Bind Chromatin to Chromatin Capture Beads:
Equilibrate the Chromatin Capture Beads to room temperature and vortex thoroughly (>30 seconds) to resuspend.
Transfer 100 μL of resuspended Chromatin Capture Beads to a new 1.5 mL tube.
Add 1,000 ng of the lysate that was stored at -80° (step 1 NOTE in Stage 2: Lysate QC, page 14) to the 1.5 mL tube containing the beads.
If the total lysate amount is <1,000 ng, add all of the lysate.
Pipette up and down to fully mix. Incubate at room temperature, off the magnetic rack, for 10 minutes.
Place the tube in the magnetic rack for 5 minutes (or until the solution looks clear).
Discard the supernatant.
Remove the tube from the magnetic rack and wash the beads with 150 μL 1X Wash Buffer.
Pipette up and down to resuspend the beads, place the tube in the magnetic rack for 1 minute and discard the supernatant.
Repeat step 6 once, for a total of 2 washes.
3.2 End polishing
Follow the steps below for End Polishing:
Remove the tube from the magnetic rack and add to the beads 53.5 μL of a master mix containing the following reagents:
| A | B | C | D | E | F | |
| Reagents | 1X | 10% extra | 3X | 5X | 8X | |
| End Polish Buffer | 50 ul | 55 ul | 165 ul | 275 ul | 440 ul | |
| End Polishing Enzyme Mix | 3.5 ul | 3.85 ul | 11.55 ul | 19.25 ul | 30.8 ul | |
| Total | 53.5 ul |
Pipette up and down to fully mix.
Incubate in an agitating thermal mixer set at 1,250 rpm as follows:
| A | B | |
| Temp | Time | |
| 22 | 30 minutes | |
| 65 | 30 minutes |
Allow the tube to reach room temperature then place it in the magnetic rack for 1 minute (or until the solution looks clear).
Discard the supernatant.
Remove the tube from the magnetic rack and wash the beads once with 150 μL 1X Wash Buffer.
Pipette up and down to resuspend the beads, place the tube in the magnetic rack.
Do not remove and discard the supernatant at this step.
Keep the tube in the magnetic rack and the beads in buffer to ensure they do not dry out while you prepare for the next reaction.
3.3 Bridge Ligation
Follow the steps below for Bridge Ligation:
Prepare and place on ice fresh 50 μL Bridge Ligation Mix by mixing the following reagents:
| A | B | C | D | E | F | |
| Reagents | 1X | 10% extra | 3X | 5X | 8X | |
| UltraPure water | 35 ul | 38.5 ul | 115.5 ul | 192.5 ul | 308 ul | |
| 5X bridge ligation buffer | 10 ul | 11 ul | 33 ul | 55 ul | 88 ul | |
| Bridge | 5 ul | 5.5 ul | 16.5 ul | 27.5 ul | 44 ul | |
| Total | 53.5 ul |
Remove and discard the supernatant from step 4 in 3.2 End Polishing, page 17.
Remove the tube from the magnetic rack and add to the beads:
| A | B | |
| Reagent | Volume/reaction | |
| Bridge ligation mix | 50 ul | |
| Bridge ligase | 1 ul | |
| Total | 51 ul |
Pipette up and down to fully mix.
Incubate at 22° for 30 minutes in an agitating thermal mixer set at 1,250 rpm.
Place the tube in the magnetic rack for 1 minute (or until the solution looks clear).
Discard the supernatant.
Remove the tube from the magnetic rack and wash the beads once with 150 μL 1X Wash Buffer.
Pipette up and down to resuspend the beads, place the tube in the magnetic rack for 1 minute and discard the supernatant.
3.4 Intra-Aggregate Ligation
Follow the steps below for Intra-Aggregate Ligation:
Remove the tube from the magnetic rack and add to the beads 52 μL of a master mix containing the following reagents:
| A | B | C | D | E | F | |
| Reagents | 1X | 10% extra | 3X | 5X | 8X | |
| Intra-Aggregate Ligation Buffer | 50 ul | 55 ul | 165 ul | 275 ul | 440 ul | |
| Intra-Aggregate Ligation enzyme mix | 2 ul | 2.2 ul | 6.6 ul | 11 ul | 17.6 ul | |
| Total | 53.5 ul |
Pipette up and down to fully mix. Incubate at 22° for 1 hour in an agitating thermal mixer set at 1,250 rpm.
SAFESTOP For convenience, this ligation reaction can proceed overnight at 22° in an agitating thermal mixer set at 1,250 rpm.
Place the tube in the magnetic rack for 1 minute (or until the solution looks clear).
Discard the supernatant.
3.5 Crosslink Reversal
Follow the steps below for Crosslink Reversal:
- Remove the tube from the magnetic rack and add to the beads 51.5 μL of a master mix containing the following reagents (in order):
| A | B | C | D | E | F | |
| Reagents | 1X | 10% extra | 3X | 5X | 8X | |
| UltraPure water | 45 ul | 49.5 ul | 148.5 ul | 247.5 ul | 396 ul | |
| 10% crosslink reversal buffer | 5 ul | 5.5 ul | 165 ul | 275 ul | 44 ul | |
| Proteinase K | 1.5 ul | 1.65 ul | 4.95 ul | 8.25 ul | 13.2 ul | |
| Total | 51.5 ul |
Pipette up and down to fully mix. Incubate in an agitating thermal mixer set at 1,250 rpm as follows:
| A | B | |
| Temp | Time | |
| 55 | 15 minutes | |
| 68 | 45 minutes | |
| 25 | hold |
SAFESTOP; For convenience, you can hold at 25° overnight in an agitating thermal mixer set at 1,250 rpm.
Place the tube in the magnetic rack for 1 minute. Transfer 50 μL of the SUPERNATANT to a new 1.5 mL tube.
Discard the beads.
3.6 DNA Purification
Follow the steps below for DNA Purification on SPRIselect Beads:
Vortex the SPRIselect beads thoroughly (>30 seconds) to resuspend.
SPRIselect reagent kitBeckman CoulterCatalog #B23317
Add 90 μL of resuspended SPRIselect beads to the 1.5 mL tube containing your sample.
Vortex to mix thoroughly.
Quick spin the tube (make sure to spin down only briefly and to stop before the beads start to settle).
Incubate the tube at room temperature, off the magnetic rack, for 5 minutes.
Quick spin the tube and place it in the magnetic rack for 5 minutes. Discard the supernatant.
Leave the tube in the magnetic rack and wash the beads twice with 200 μL fresh 80% ethanol.
Do not resuspend the beads for these washes.
Simply add the ethanol, wait for 1 minute then discard the ethanol supernatant.
After the last wash, quick spin the tube and place it in the magnetic rack for 1 minute.
Use a 10 μL pipette tip to remove traces of ethanol
Air dry the beads for 5 minutes in the magnetic rack until no residual ethanol remains.
Do not over dry the beads.
Off the magnetic rack, resuspend the beads in 52 μL TE Buffer pH 8.0.
Vortex to mix thoroughly.
Quick spin the tube (make sure to spin down only briefly and to stop before the beads start to settle).
Incubate at room temperature, off the magnetic rack, for 5 minutes.
Quick spin the tube and place it in the magnetic rack for 1 minute.
Transfer 50 μL of the SUPERNATANT (purified DNA) to a new tube. Discard the beads.
Quantify the purified DNA using a Qubit Fluorometer and Qubit dsDNA HS Kit.
You should recover a minimum of 50 ng to proceed to Step 4: Library Preparation
You will use 150 ng of your purified DNA for Stage 4 in a 50 μL volume. If needed, you can bring up the volume to 50 μL using TE Buffer pH 8.0.
If you recovered <150 ng, use all of the purified DNA to proceed to Stage 4.
If you recovered >150 ng, use 150 ng to proceed to Stage 4 and keep the remaining purified DNA stored at -20° You can use the remaining
SAFESTOP ; Purified DNA sample can be stored at -20° for up to 6 months.
pre-step 4
Stage 4: Library Preparation
As you prepare for Stage 4, keep the following in mind:
The library preparation protocol does not require fragmentation.
The library preparation protocol takes ~ 2.5 hours.
Dovetail Hi-C Library Preparation KitCatalog #21004
Before You Begin
The End Repair Buffer may have precipitated in storage.
Incubate for at least 10 minutes at 37° until there is no visible precipitate.
Equilibrate TE Buffer pH 8.0 and SPRIselect beads to room temperature.
Thaw 250 mM DTT and Adaptor for Illumina at room temperature.
Vortex to mix prior to use.
Step 4
4.1 End Repair
Follow the steps below for End Repair:
Place 50 μL of purified DNA (150 ng) in a 0.2 mL PCR tube.
Add to the PCR tube 10.5 μL of a master mix containing the following reagents:
| A | B | C | D | E | F | |
| Reagents | 1X | 10% extra | 3X | 5X | 8X | |
| End Repair Buffer | 7 ul | 7.7 ul | 23.1 ul | 38.5 ul | 61.6 ul | |
| End repair enzyme mix | 3 ul | 3.3 ul | 9.9 ul | 16.5 ul | 26.4 ul | |
| 250 mM DTT | 0.5 ul | 0.55 ul | 1.65 ul | 2.75 ul | 4.4 ul | |
| Total | 10.5 ul |
Pipette up and down to fully mix.
Quick spin the tube.
Incubate in a thermal cycler as follows:
| A | B | |
| Temp | Time | |
| 20 | 30 minutes | |
| 65 | 30 minutes | |
| 12 | hold |
4.2 Adapter Ligation and USER Digest
Follow the steps below for Adapter Ligation and USER Digest:
Add to the PCR tube containing the end-repaired sample the following reagents:
| A | B | |
| Reagent | Volume/reaction | |
| Adaptor for Illumina | 2.5 ul | |
| Ligation Enzyme mix | 30 ul | |
| Ligation Enhancer | 1 ul | |
| Total | 33.5 ul |
NOTE The Ligation Enzyme Mix and Ligation Enhancer can be mixed ahead of time.
The master mix is stable for 8 hours at 4°
We do not recommend adding the Adaptor for Illumina to the master mix.
Pipette up and down to fully mix.
Quick spin the tube.
Incubate at 20° for 15 minutes in a thermal cycler.
Hold at 12°
Following incubation, add 3 μL of USER Enzyme Mix to the PCR tube.
Pipette up and down to fully mix.
Quick spin the tube.
Incubate at 37 ° for 15 minutes in a thermal cycler.
Hold at 12 °
4.3 DNA Purification
Follow the steps below for DNA Purification:
Vortex the speed beads thoroughly (>30 seconds) to resuspend.
Add 80 μL of resuspended speed beads to the PCR tube containing the adaptor-ligated sample.
Vortex to mix thoroughly.
Quick spin the tube (make sure to spin down only briefly and to stop before the beads start to settle).
Incubate the tube at room temperature, off the magnetic rack, for 5 minutes.
Quick spin the tube and place it in the magnetic rack for 5 minutes. Discard the supernatant.
Leave the tube in the magnetic rack and wash the beads twice with 200 μL fresh 80% ethanol.
Do not resuspend the beads for these washes.
Simply add the ethanol, wait for 1 minute then discard the ethanol supernatant.
Quick spin the tube and place it in the magnetic rack for 1 minute. Use a 10 μL pipette tip to remove traces of ethanol
Air dry the beads for 5 minutes in the magnetic rack until no residual ethanol remains. Do not over dry the beads.
Off the magnetic rack, resuspend the beads in 100 μL TE Buffer pH 8.0.
Vortex to mix thoroughly.
Quick spin the tube (make sure to spin down only briefly and to stop before the beads start to settle).
Incubate at room temperature, off the magnetic rack, for 5 minutes.
Quick spin the tube and place it in the magnetic rack for 1 minute.
Transfer 95 μL of the SUPERNATANT (purified adaptor-ligated DNA) to a new tube. Discard the beads.
SAFESTOP Purified DNA sample can be stored at -20° overnight.
Pre-Step 5
Stage 5: Ligation Capture and Amplification
As you prepare for Stage 5, keep the following in mind:
The Ligation Capture and Amplification protocol takes ~ 2 hours.
Dovetail Hi-C Library Preparation KitCatalog #21004
Before You Begin
Thaw Universal PCR Primer, Index Primer, and HotStart PCR Ready Mix at room
temperature.
Vortex to mix prior to use.
Equilibrate TE Buffer pH 8.0, speed beads, Streptavidin Beads, TWB, 2X NTB, LWB, and NWB to room temperature.
Step 5
5.1 Streptavidin Beads Preparation
NOTE This step does not involve any DNA sample.
Follow the steps below for Ligation Capture and Amplification:
Vortex the Streptavidin Beads vial thoroughly (> 30 seconds) to resuspend the beads.
Transfer 25 μL of resuspended Streptavidin beads to a new 1.5 mL tube.
Place the 1.5 mL tube containing the beads in the magnetic rack for 5 minutes.
Discard the supernatant.
Remove the tube from the magnetic rack and wash the beads with 200 μL TWB: pipette up and down to resuspend the beads and place the tube in the magnetic rack for 1 minute.
Discard the supernatant.
Repeat step 3 once, for a total of 2 washes.
After the second wash, resuspend the beads in 100 μL 2X NTB.
Pipette up and down to fully mix.
5.2 Ligation Capture
Follow the steps below for Ligation Capture:
Transfer the 95 μL of purified adaptor-ligated DNA (from step 13 in 4.3 DNA Purification, page 24) to the 1.5 mL tube containing the Streptavidin beads resuspended in 100 μL of 2X NTB.
Vortex to mix thoroughly. Quick spin the tube (make sure to spin down only briefly and to stop before the beads start to settle).
Incubate at 25 ̊ for 30 minutes in an agitating thermal mixer set at 1,250 rpm.
5.3 Wash Sample on Streptavidin Beads
NOTE For each of the washes below, remove the tube from the magnetic rack, add the indicated buffer to the beads, pipette up and down to resuspend the beads, place the tube in the magnetic rack for 1 minute, and discard the supernatant. Remove all of the supernatant between each wash; residual supernatant can interfere with the downstream PCR.
Follow the steps below for Wash Sample on Streptavidin Beads:
Quick spin the tube and place it in the magnetic rack for 1 minute. Discard the supernatant.
Wash the beads once with 200 μL LWB.
Wash the beads twice with 200 μL NWB.
Wash the beads twice with 200 μL 1X Wash Buffer.
5.4 Index PCR
NOTE Not all PCR enzymes and master mixes are compatible for amplification in the presence of Streptavidin beads. Please use the HotStart PCR Ready Mix supplied in your Dovetail Kit (Box 2).
Follow the steps below for Index PCR:
After the last wash, remove the tube from the magnetic rack and add to the beads 45 μL of a master mix containing the following reagents:
| A | B | C | D | E | F | |
| Reagents | 1X | 10% extra | 3X | 5X | 8X | |
| UltraPure water | 15 ul | 16.5 ul | 49.5 ul | 82.5 ul | 132 ul | |
| HotStart PCR Ready Mix | 25 ul | 27.5 ul | 82.5 ul | 137.5 ul | 220 ul | |
| Universal PCR primer | 5 ul | 5.5 ul | 16.5 ul | 27.5 ul | 44 ul | |
| Total | 45 ul |
Add 5 μL Index Primer to the PCR reaction.
Use one Index Primer per PCR reaction
Pipette up and down to fully mix then transfer to a new 0.2 mL PCR tube.
Quick spin the tube (make sure to spin down only briefly and to stop before the beads start to settle).
Place the tube into the thermal cycler and run the following program:
| A | B | C | D | |
| Step | Temp | Time | Cycles | |
| Enzyme activation | 98 | 3 minutes | 1 | |
| Denature | 98 | 20 seconds | 12 | |
| Anneal | 65 | 30 seconds | ||
| Extend | 72 | 30 seconds | ||
| Extend | 72 | 1 minute | 1 | |
| 12 | hold | 1 |
5.5 Size Selection
Sera-Mag SpeedBeads Carboxylate-Modified Magnetic ParticlesGE HealthcareCatalog #44152105050350
Follow the steps below for Size Selection:
Quick spin the PCR tube and place it in the magnetic rack for 1 minute.
Transfer 47 μL of the SUPERNATANT to a new 1.5 mL tube.
Discard the beads.
Add 53 μL of TE Buffer pH 8.0 to the 1.5 mL tube to bring the volume of the sample in the tube to 100 μL.
Vortex the speed beads thoroughly (>30 seconds) to resuspend.
Add 50 μL of resuspended speed beads to the 1.5 mL tube containing your sample.Sera-Mag SpeedBeads Carboxylate-Modified Magnetic ParticlesGE HealthcareCatalog #44152105050350
Vortex to mix thoroughly. Quick spin the tube (make sure to spin down only briefly and to stop before the beads start to settle).
Incubate the tube at room temperature, off the magnetic rack, for 10 minutes.
Quick spin the tube and place it in the magnetic rack for 5 minutes.
Transfer 145 μL of the SUPERNATANT to a new 1.5 mL tube. Discard the beads.
Add 30 μL of resuspended Speed beads to the 1.5 mL tube containing your sample.Sera-Mag SpeedBeads Carboxylate-Modified Magnetic ParticlesGE HealthcareCatalog #44152105050350
Vortex to mix thoroughly.
Quick spin the tube (make sure to spin down only briefly and to stop before the beads start to settle).
Incubate the tube at room temperature, off the magnetic rack, for 10 minutes.
Quick spin the tube and place it in the magnetic rack for 5 minutes.
Discard the supernatant.
Leave the tube in the magnetic rack and wash the beads twice with 200 μL fresh 80% ethanol.
Do not resuspend the beads for these washes.
Simply add the ethanol, wait for 1 minute then discard the ethanol supernatant.
Quick spin the tube and place it in the magnetic rack for 1 minute. Use a 10 μL pipette tip to remove traces of ethanol.
Air dry the beads for 5 minutes in the magnetic rack until no residual ethanol remains. Do not over dry the beads.
Off the magnetic rack, resuspend the beads in 30 μL TE Buffer pH 8.0.
Vortex to mix thoroughly.
Quick spin the tube (make sure to spin down only briefly and to stop before the beads start to settle).
Incubate the tube at room temperature, off the magnetic rack, for 5 minutes.
Quick spin the tube and place it in the magnetic rack for 1 minute.
Transfer 28 μL of the SUPERNATANT to a new 1.5 mL tube. The supernatant is your size selected library. Discard the beads.
Quantify your size selected library using a Qubit Fluorometer and Qubit dsDNA HS Kit.
Use a TapeStation or Bioanalyzer to verify the size distribution of your size selected library.
The size range is expected to be between 350 bp and 1,000 bp.
SAFESTOP The library can be stored at -20° for up to 6 months.