Jun 30, 2025

Public workspacesortChIC in bulk [version without A-tailing]

Nature Cell Biology
DOI
https://dx.doi.org/10.17504/protocols.io.j8nlkd4odg5r/v1
  • Samy Kefalopoulou1,
  • Peter Zeller1,2
  • 1Hubrecht Institute for Developmental Biology and Stem Cell Research;
  • 2Aarhus University, Department of Molecular Biology and Genetics
  • Dam&ChIC
Samy Kefalopoulou
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DOI: https://dx.doi.org/10.17504/protocols.io.j8nlkd4odg5r/v1
Protocol Citation: Samy Kefalopoulou, Peter Zeller 2025. sortChIC in bulk [version without A-tailing]. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlkd4odg5r/v1
Manuscript citation:
Zeller, P. et al. Single-cell sortChIC identifies hierarchical chromatin dynamics during hematopoiesis. Nat Genet 55, 333–345 (2023).
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: April 07, 2025
Last Modified: June 30, 2025
Protocol Integer ID: 126297
Keywords: ChIC, chromatin, bulk methods, chromatin profiling, original bulk sortchic protocol, cell sortchic, bulk sortchic, present version of bulk sortchic, sortchic in bulk, crude bulks of cell, sortchic, cell experiment, antibodies in bulk, specialized robotic liquid handler, bulk chic dataset against histone h3, robotic liquid handler, bulk chic dataset, sorted population, cell, normalization control for the chic readout, nuclei quality, cell population, chic readout, bulk, crude bulk, cell version, populations of cell
Abstract
Bulk sortChIC is an adaptation of single-cell sortChIC (Zeller et al., 2023, Gaza et al., 2024) tailored to sorted populations of cells (e.g. 20c-10000c). The protocol doesn't require the specialized robotic liquid handlers used in its single-cell version. Ideally, cell populations are sorted by FACS to ensure good cell/nuclei quality and selection based on cell-cycle state. If that is not possible, the protocol could also be performed on crude bulks of cells as long as cell/nuclei quality is confirmed to be high.

In the present version of bulk sortChIC, we omit A-tailing and do ligation/barcoding with blunt-end adapters.
This adaptation was made to enable direct comparisons of the different readouts during development of Dam&ChIC. It might be a useful protocol if one has available blunt-end adapters. In any other case, we recommend following the original bulk sortChIC protocol described by Zeller et al., 2023 and Gaza et al., 2024, which includes A-tailing and ligation with T-tailed adapters.

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We used the present adaptation in the Dam&ChIC manuscript (Kefalopoulou et al., 2025, Figure 1/ED1) to generate a bulk ChIC dataset against histone H3, serving as normalization control for the ChIC readout of Dam&ChIC. We also extensively used this adaptation to titrate antibodies in bulk (100c populations) prior to use in single-cell experiments.
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Materials
For a full list of materials used in the original bulk sortChIC protocol check also Zeller et al., 2023 and Gaza et al., 2024.

Here we provide materials relevant to this modified version:

Antibodies
●      Rabbit polyclonal anti-Lamin B1 (Abcam, ab16048)
●      Rabbit monoclonal anti-H3K27me3 (Cell Signaling Technologies, 9733S)
●      Rabbit monoclonal anti-H3K9me3 RM389 (Thermofisher, MA5-33395)
●      Rabbit monoclonal anti-H3K4me3 (Thermofisher, MA5-11199)
●      Rabbit monoclonal anti-Histone H3 (Abcam, ab176842)
●      Rabbit monoclonal anti-H2AK119Ub (Cell Signaling, D27C4)

 
Oligonucleotides
●      Adapters for DamID2, top and bottom oligonucleotides, set of 384
Download DamID2_adapters_384_top_bottom_annotated.xlsxDamID2_adapters_384_top_bottom_annotated.xlsx36KB
●     Adapters for DamID2, top and bottom oligonucleotides, set of 96
Download DamID2_adapters_96_top_bottom_annotated.xlsxDamID2_adapters_96_top_bottom_annotated.xlsx15KB

●      Random hexRT primer
GCCTTGGCACCCGAGAATTCCANNNNNN

●      Illumina RNA PCR primer 1 (RP1)
●      Illumina RNA PCR index primers (RPI series)
Download RPI_primers.xlsxRPI_primers.xlsx10KB
 

Chemicals and Buffers
For sample preparation:
●      PBS0
●      Ethanol
●      Saponin (Sigma, 47036-50G-F)
●      Tween 20 (Sigma, P9416)
●      cOmplete Mini EDTA-free Protease Inhibitor Cocktail (Roche, 11836170001)
●      HEPES 1M (Gibco, 15630080)
●      Spermidine Solution (Sigma, S2626-1G)
●      Sodium Chloride (NaCl) 5M
●      EDTA 0.5M
●      Hoechst 34580 (Sigma-Aldrich, 63493-5MG)

For CellTrace stainings:
●      CellTraceTM CFSE Cell Proliferation kit (Invitrogen, C34570)
●      CellTraceTM Far Red Cell Proliferation kit (Invitrogen, C34572)
●      CellTraceTM Yellow Cell Proliferation kit (Invitrogen, C34573)
●      Rat Serum (Sigma, R9759)
●      DMSO

For molecular processing:
●      Calcium Chloride (CaCl2) 1M
●      EGTA 0.5M 
●      Igepal CA-630 (Sigma, I8896-50ML)
●      DTT 1M (Invitrogen, Y00147)
●      10X PNK buffer (NEB, B0201S)
●      dNTP set 100mM (Invitrogen, 10297018)
●      Magnesium Chloride (MgCl2) 25mM (NEB, B9021S)
●      ATP 10mM (NEB, P0756L)
●      BSA Molecular Biology Grade 20 mg/ml (NEB, B9000S)
●      PEG8000 50% (Promega, V3010)
●      Mineral Oil (Sigma-Aldrich, M8410)
●      Bead-binding buffer (1M NaCl, 20% PEG8000, 20mM Tris-HCl pH = 8, 1mM EDTA)
●      Fragmentation buffer (500mM potassium acetate, 150mM magnesium acetate, 200mM Tris-acetate)
●      CleanNGS DNA and RNA purification beads (GC Biotech, CNGS-0050)


Enzymes
●      PA-MNase fusion recombinant protein, self-produced
●      Proteinase K solution 20 mg/ml (Ambion, AM2548)
●      DNA polymerase I, Large (Klenow) Fragment (NEB, M0210L)
●      T4 Polynucleotide Kinase (NEB, M0201L)
●      T4 DNA Ligase (Roche, DNALIG-RO)
●      Superscript II Reverse Transcriptase (Thermofisher, 18064071)
●      RNAseOUT Recombinant Ribonuclease Inhibitor (Invitrogen, 10777019)
●      Phusion High Fidelity 2X PCR mastermix (NEB)


Commercial Assays
●      MEGAscript T7 transcription kit (Invitrogen, AMB13345)
●      Agilent High Sensitivity DNA Assay (Agilent, 5067-4626)
●      Agilent RNA 6000 Pico Assay (Agilent, 5067-1513)
●      Qubit sdDNA High Sensitivity Assay (Invitrogen, Q32854)
 

Other 
●      Protein LoBind tubes 0.5 ml (Eppendorf, 0030108094)
●      Low-retention pipette tips (Greiner Bio-One)
●      Polypropylene round bottom tubes 5 ml (Corning, 352002)
●      Qubit 4 fluorometer (Invitrogen)
●      Agilent 2100 Bioanalyzer platform


FACS
●      BD FACS Influx Cell Sorter System
●      BD FACS Jazz Cell Sorter System
 

Sequencing
●      Illumina NextSeq500 sequencing platform
●      Illumina NextSeq2000 sequencing platform
Troubleshooting
Before start
NOTES on handling and equipment during the protocol
  • Upon permeabilization or fixation, samples should be kept at 4oC, and whenever washes are involved, they should be done in a cooled centrifuge.
  • To increase nuclei or fixed cell recovery after washes, we recommend the use of a swing-out cooled centrifuge instead of conventional one.
  • Use low-binding tubes and low-retention pipette tips at all times when handling nuclei or fixed cells, in order to prevent losses.
  • With every spin and discarding supernatant of nuclei or fixed cells, make sure to pipette carefully without disturbing the pellet. We do not recommend aspirating with a vacuum.
  • This protocol makes use of self-produced pA-MNase. Targeting of the protein may need further optimizations when using different batches or a commercial enzyme.
  • Ensure that both bench and pipettes are decontaminated from RNases and DNases when handling pre-amplified material, and especially when handling amplified RNA.
  • Make sure you are familiar with the process and principles of DNA/RNA cleanups using beads (eg Ampure/SPRI), as they are used extensively in this protocol.


Preparation
Preparation of ChIC buffers

Wash buffer (WB0): the basic ChIC buffer for nuclei
solutionvolumeconcentration in ChIC buffer
Ultra-pure water47.5 mL
1M HEPES pH 7.51 mL20 mM
5M NaCl1.5 mL150 mM
pure spermidine solution3.6 uL66.6 ug/ml
10% Saponin250 uL0.05%


Wash buffer 0f (WB0f): the basic ChIC buffer for fixed cells
solutionvolumeconcentration in ChIC buffer
Ultra-pure water47.5 mL
1M HEPES pH 7.51 mL20 mM
5M NaCl1.5 mL150 mM
pure spermidine solution3.6 uL66.6 ug/ml
10% Tween-20250 uL0.05%

Wash buffer 1 (WB1): the antibody incubation buffer for nuclei
WB0 + protease inhibitors + 4 uL/mL 0.5M EDTA
Wash buffer 1f (WB1f)*: the antibody incubation buffer for fixed cells
WB0f + protease inhibitors + 4 uL/mL 0.5M EDTA
*a variation of WB1f is used specifically during Cell-Trace stainings of fixed cells, in which the spermidine is omitted

Wash buffer 2 (WB2): the pA-MNase incubation buffer for nuclei
WB0 + protease inhibitors
Wash buffer 2f (WB2f): the pA-MNase incubation buffer for fixed cells
WB0f + protease inhibitors

Activation solution: the buffer used to activate pA-MNase
WB0 or WB0f containing 4 mM CaCl2

STOP solution: the buffer used to stop pA-MNase activity and lyse the cells
67 uL nuclease-free water
8 uL 0.5M EGTA
15 uL 10% NP40
10 uL ProtK (Ambion, AM2548)

NOTES:
  • Saponin and Tween 10% stock solutions should be put on a roller to dissolve properly.
  • Saponin solution should be always made fresh, Tween can be aliquoted and frozen at -20oC.
  • ChIC buffers are preferably made fresh and used within 24 hours. Buffers made for the overnight antibody staining can be stored at 4oC and used the following day.


Preparation of sorting tubes
Prior to the sorting populations by FACS, prepare PCR strips or 0.5mL tubes with 5 uL WB0 or WB0f.

Optionally, the tubes can also contain 10-50 uL mineral oil, similarly to the 384-well plates in the single-cell sortChIC protocol, but it is not necessary in our experience.

NOTE:
If no mineral oil is used, make sure all incubations in PCR machines are done with heated lid.


Sample preparation for antibody staining
Harvest cells and Wash
  1. Harvest cells and make a nice single-cell suspension.
  2. Wash cells two-three times with room-temperature PBS0 (without Ca2+ and Mg2+)
  3. Count on a cytometer.

There are two alternative downstream steps:
(i) To permeabilize the cells and isolate nuclei in their native condition, proceed to step 7.
(ii) To fix the cells with 70% Ethanol, then permeabilize, proceed to step 8.

Fixation gives the advantage of long-term sample storage, particularly useful for samples that are challenging to obtain (e.g. from a differentiation time-course)


Alternative 1: Nuclei isolation
  1. Depending on your cell count, split the sample into multiple 0.5mL low-binding tubes if multiple stainings will be done. Use around 0.5 million cells per antibody staining.
  2. Spin down at 300rcf for 4 minutes and resuspend in 400 uL Wash Buffer 1 (WB1) per tube/staining, while keeping the sample at 4oC. Proceed to step 6.


Alternative 2: Cell fixation with Ethanol
  1. Pre-cool 100% Ethanol, by placing it at -20oC a few hours beforehand.
  2. Resuspend 1 million cells in 300 uL ice-cold PBS0 in a 15mL tube (ideally in a low-binding tube to prevent cell loss)
  3. While vortexing, add drop-by-drop 700 uL ice-cold 100% Ethanol.
  4. Fix for 1-2 hours at -20oC. It is also possible to fix overnight.
  5. Spin tubes at 300rcf for 4 minutes in a cooled centrifuge (4oC) and remove supernatant. If none of the optional following steps are desired, proceed to step 7.

NOTES:
  • It is important that fixation is done properly and drop-wise in order to avoid formation of clumps.
  • Scale the volume accordingly if cell numbers are higher or lower. E.g. fix 0.5 million cells in 500uL total volume, 2 million cells in 2 mL total volume etc.


(optional)
Cell-Trace stainings for sample multiplexing
To enable parallel processing of multiple samples/conditions, and thereby minimize batch effects, cells can be stained with CellTrace dyes. Samples stained uniquely with CellTrace dyes can be mixed together right before the antibody staining step and their respective populations will be distinguished during FACS.

  1. Resuspend cells in Wash Buffer 1f (WB1f), in which no spermidine is added.
  2. Transfer cells in low-binding 1.5mL tubes.
  3. Wash cells once more with WB1f (-sperm).
  4. Stain 1 million cells in 1 mL WB1f (-sperm) with 0.25 uL Cell-Trace dye. If necessary to stain with multiple non-overlapping dyes, add 0.25 uL of each.
  5. Incubate for 20-30 minutes at 4oC protected from light.
  6. Quench the staining with the addition of 50 uL rat serum.
  7. Incubate for 10 minutes at 4oC protected from light.
  8. Spin down at 300rcf for 4 minutes at 4oC. Remove supernatant.

NOTES:
  • Two washes are required to remove as much EtOH as possible prior to stainings.
  • We have successfully used Cell-tracer dyes CFSE (C34570, Invitrogen), Yellow (C34573, Invitrogen) and Far-Red (C34572, Invitrogen), and combinations of them, to multiplex up to 8 different samples together.
  • The use of other colors is possible but they should not overlap with Hoechst 34580, which will be used to measure DNA content during FACS.
  • Cells stained with Cell-Trace dyes can be cryopreserved (step 5.2), or immediately used for antibody staining (step 7)


(optional)
Cryopreservation of EtOH-fixed samples
  1. Resuspend cells in WB1f.
  2. Spin down at 300rcf for 4 minutes at 4oC. Remove supernatant.
  3. Repeat with a 2nd wash in WB1f.
  4. If desired, make aliquots of cells in 0.5mL low-binding tubes.
  5. Add the same volume of WB1f containing 20% DMSO (final 10% DMSO per sample)
  6. Freeze at -80oC. Cryopreservation is long-term and samples can remain in good quality for several years.

NOTE:
  • If Cell-Trace stainings were done prior to cryopreservation, it is handy to make aliquots of 100-200K cells per staining. Plan this accordingly to how many samples will be mixed and stained together for sorting.


Antibody staining
Antibody staining of nuclei
At this point each sample is resuspended in 400uL WB1.
  1. Spin down at 300rcf for 4 minutes at 4oC. Remove supernatant.
  2. Resuspend in 400 uL of WB1 containing the primary antibody.
  3. Mix overnight at 4oC on a roller.


Antibody staining of fixed cells

For cryopreserved EtOH-fixed cells:
  1. Thaw cells from -80oC
  2. Spin down at 300rcf for 4 minutes at 4oC
  3. Wash cells twice with WB1f to remove DMSO.
  4. Resuspend in 400 uL of WB1f containing the primary antibody.
  5. Incubate overnight at 4oC on a roller.


For multiplexing of cryopreserved EtOH-fixed Cell-Tracer stained cells:
  1. Thaw cells from -80oC
  2. Mix together the different populations in equal cell numbers in a 0.5mL or 1mL low-binding tube to make a "super-sample". E.g. 200K cells stained with CT CFSE + 200K cells stained with CT Far-Red + 200K cells stained with CT Yellow
  3. Spin down at 300rcf for 4 minutes at 4oC
  4. Wash once with WB1f to remove DMSO.
  5. Transfer to a 0.5mL low-binding tube (optional) and repeat wash with WB1f.
  6. Resuspend in 400 uL of WB1f containing the primary antibody.
  7. Incubate overnight at 4oC on a roller.


For cells freshly-fixed with EtOH:
At this point EtOH-fixed cells are in a 15ml tube, spun down once and supernatant is removed.
  1. Resuspend cells in WB1f.
  2. Transfer 0.5 million cells to a 0.5mL low-binding tube
  3. Wash cells twice with WB1f to remove all EtOH.
  4. Resuspend in 400 uL of WB1f containing the primary antibody.
  5. Incubate overnight at 4oC on a roller.


For cells freshly-fixed with EtOH and stained with Cell Tracers:
  1. Resuspend cells in WB1f.
  2. Mix together the different populations in equal cell numbers in a 0.5mL or 1mL low-binding tube to make a "super-sample". E.g. 200K cells stained with CT CFSE + 200K cells stained with CT Far-Red + 200K cells stained with CT Yellow
  3. Spin down at 300rcf for 4 minutes at 4oC
  4. Remove supernatant.
  5. Transfer to a 0.5mL low-binding tube (optional) and repeat wash with WB1f.
  6. Resuspend in 400 uL of WB1f containing the primary antibody.
  7. Incubate overnight at 4oC on a roller.



NOTES:
  • The ideal amount of primary antibody to be used for stainings differs per antibody (even per LOT number for polyclonal antibodies) and should be titrated to ensure proper enrichment and low signal to noise ratio.
  • We recommend testing a series of concentrations around the recommended by the manufacturer, e.g. 1:200, 1:400, 1:1000 etc using bulk sortChIC, as described in the related protocol here or previously by Zeller et al., 2023 or Gaza et al., 2024.
  • pA-MNase has high affinity for rabbit IgG. If the primary antibody is not raised in rabbit, a secondary antibody staining has to be done for 1 hour at 4oC, right before pA-MNase tethering (step 9).


pA-MNase tethering
pA-MNase tethering and parallel staining for DNA content
  1. Spin the nuclei or fixed cells at 300 rcf for 4 minutes at 4oC.
  2. Resuspend the pellet with Wash Buffer 2 (WB2) for nuclei, or Wash Buffer 2f (WB2f) for fixed cells.
  3. Spin again at 300 rcf for 4 minutes at 4oC.
  4. Resuspend nuclei in 500 uL WB2 or WB2f containing pA-MNase.
  5. In the same mix add Hoechst 34580 at a final concentration of 2.5 ug/mL.
  6. Incubate for 1 hour at 4oC on roller

NOTES:
  • The ideal amount of pA-MNase depends on the batch of protein production (for self-produced protein) and on the cell type.
  • In the Dam&ChIC manuscript, KBM7 samples (data in Figures 1, 2 and 3) were incubated with pA-MNase at a final concentration of 3 ng/uL (1:200), while mouse ESCs and Vitamin C samples at a final concentration of 0.6 ng/uL (1:1000).


Washes and transfer to FACS tubes
  1. Wash the nuclei twice with WB2 or WB2f, like above.
  2. After the final wash, resuspend in 600 uL WB2 or WB2f.
  3. Pipette the suspension in a FACS tube through a filter cap to get rid of clumps.


Bulk sort (FACS)
  1. Analyze samples by FACS to set the desired gates. See example strategy below.
  2. Sort the desired cell number (e.g. 100, 1000, 10000 cells) in PCR strips or 0.5mL tubes prefilled with 5 uL WB0 or WB0f (step 2). Sorting should be done on cooling settings.
  3. After sorting of each strip or tube is complete, perform a quick spin and keep samples on cool blocks at 4oC at all times.


Digestion, end-processing and barcoding
pA-MNase activation
  1. Add 5 uL of Activation solution (step 1) per bulk sample, while keeping the samples at 4oC.
  2. Do a quick spin.
  3. Digest for exactly 30 minutes at 4oC.

NOTES:
  • It is important to precisely time the activation of pA-MNase and keep it constant across samples, especially if they represent technical replicates.


Proteinase K treatment and Lysis
Stop digestion by adding 10 uL of Stop Solution (step 1) to each bulk sample.
The total volume at this point is 20 uL.

Incubate with the following program in a PCR machine:
65oC for 6 hours
80oC for 20 minutes
Hold at 4oC

Samples can be frozen at -20oC for long-term storage.


End-repair of fragments produced by pA-MNase

Add 10 uL of Blunt-ending mix to each bulk sample:
ReagentVolume (uL) per sample
Klenow Large 3'-5' exo (5000 U/uL)0.2
dNTPs (10 mM)0.5
T4 PNK (10000 U/uL)0.2
ATP (10 mM)3
PNK buffer 10X3
MgCl2 (25 mM)1
PEG80000.5
BSA (20 mg/mL)0.3
H2O1.3
TOTAL10
Cumulative volume30
Incubate with the following program in a PCR machine:
37oC for 30 minutes
75oC for 20 minutes
Hold at 4oC

NOTE:
  • During the end-repair program we recommend moving the plates on a cool block at 4oC directly when 37oC incubation is finished, and move them back to the PCR machine once the block reaches 75oC.


Barcoding/Adapter ligation

Add 2 uL of a unique DamID2 adapter per bulk sample
(final adapter concentration can be in the range of 20-50nM)

Add 48 uL of Ligation mix to each bulk sample:
ReagentVolume (uL) per sample
T4 ligase (5U/uL)1
T4 ligase buffer 10X8
H2O39
TOTAL48
FINAL cumulative volume80
Incubate with the following program in a PCR machine:
4oC for 20 minutes
16oC for 16 hours
65oC for 10 minutes
Hold at 4oC

NOTE:
We provide both the set of 384 and the set of 96 DamID2 adapter sequences in Materials, and either of them can be used for bulk experiments. We prefer using the set of 96, so to keep reagents for single-cell and bulk experiments separate.


Pooling and amplification
Pooling
Pool uniquely-barcoded bulk samples in one eppendorf tube for downstream processing.

NOTES:
  • We especially recommend pooling when handling technical replicates and samples for titrations of antibodies. In general, it makes the process more efficient and limits variation/batch effects during downstream steps.
  • Do a couple of transfers to clean tubes, by leaving behind the oil phase in case the samples were sorted in tubes containing mineral oil.


DNA purification
  1. Per sample, add 0.8 volume beads diluted 1:10 in bead-binding buffer (see recipe in Materials).
  2. Incubate for 20-30 minutes at room temperature.
  3. Put samples on magnetic stand and incubate until all beads are bound to the magnet (liquid should look clear, around 20 minutes).
  4. Remove unbound liquid.
  5. Wash three times with freshly-made 80% ethanol.
  6. During the last wash with ethanol, use a stronger hand magnet to concentrate the beads as much as possible at one place in the tube (they tend to be dispensed across the length of the tube)
  7. Remove ethanol and let the beads air-dry until they look matte.
  8. Elute in 6 uL ultra-pure water (DNase/RNase-free) for 10 minutes.
  9. Transfer the eluted samples to PCR strips for the IVT reaction (step 18)

NOTES:
  • It is important that the beads don't over-dry before elution, as this can result in irreversible binding of fragments.
  • It is not necessary to separate the eluted material from the beads for the IVT reaction that follows.


Linear amplification by in vitro transcription (IVT)
Per sample add 9 uL of IVT mix, according to manufacturer instructions (MEGAScript T7 transcription kit):
ReagentVolume (uL)
A1.5
U1.5
G1.5
C1.5
T7 buffer1.5
T7 enzyme1.5
Total reaction volume is 15 uL

Incubate with the following program in a PCR machine:
37oC for 14 hours
Hold at 4oC

The amplified RNA (aRNA) can be stored long-term at -80oC.


aRNA purification
  1. Measure the exact volume of each aRNA sample with a pipette (should be around 15 uL) and transfer to a 1.5 mL tube.
  2. Add ultra-pure water up to 30 uL.
  3. Add 0.8 volume of undiluted beads. Incubate for 10 minutes.
  4. Put samples on a magnetic stand and incubate until all beads are bound to the magnet (liquid should look clear).
  5. Remove unbound liquid.
  6. Wash three times with freshly-made 80% ethanol.
  7. Air-dry beads until they look matte.
  8. Elute in 13 uL ultra-pure water. Leave for 10 minutes.
  9. Put on magnetic stand until clear and transfer the eluate in a clean tube.


(optional)
aRNA fragmentation
  1. Add ultra-pure water up to 20 uL.
  2. Add 0.2 volume fragmentation buffer.
  3. Incubate in a pre-heated block at 94oC for 90-120 seconds.
  4. Transfer on ice and stop fragmentation with 0.1 volume 0.5M EDTA.

Repeat purification of the aRNA, like in step 19.


aRNA quantification
  1. Measure 1 uL of aRNA on the Nanodrop to estimate total amount of product. In case this measurement gives a total yield of higher than 1 ug per 100c population sample, we know empirically that the sample contains too much free adapter and most likely requires extra clean-ups. In that case, repeat step 19 and measure on Nanodrop again.
  2. Run 1 uL of aRNA on the Bioanalyzer (Total Eukaryote RNA Assay) or equivalent.

NOTES:
  • Based on the size distribution of the product, determine the ratio between aRNA and free adapter. Empirically, we know that if the adapter peak is more than two to three times as big as the aRNA product, it is necessary to perform extra bead clean-ups (1 to 3 extra), to remove as much as possible of the adapters. This is important as high amount of adapters may be amplified during library preparation and negatively affect the sequencing.
  • Keep in mind that extra bead clean-up may entail some loss of product, so the decision whether and how many extra to perform should also depend on the amount of actual aRNA product you see on the Bioanalyzer at this first quantification.
  • In case more bead clean-ups are needed, repeat step 19 and make sure to do a quantification of the final aRNA product, before proceeding with library preparation.


Library preparation
Reverse transcription
  1. Take 100ng of aRNA product diluted in 5 uL ultra-pure water. This amount can definitely be lower, in case the aRNA product is not abundant.
  2. Add a mix of 1 uL Random Hexamer primer (20 uM) + 0.5 uL dNTPs (10mM).
  3. Incubate @65oC for exactly 5 minutes.
  4. Quickly transfer samples on ice.
  5. Add 4 uL of RT mix:
ReagentVolume (uL)
5X First-Strand buffer2
DTT 0.1M1
RNAse OUT0.5
Superscript II0.5
Total reaction volume is 10.5 uL

Incubate with the following program in a PCR machine:
25oC for 10 minutes
42oC for 1 hour
Hold at 4oC

NOTE:
  • The Random Hexamer primer sequence is GCCTTGGCACCCGAGAATTCCANNNNNN (Markodimitraki et al., 2020) and it includes the Illumina P7. Check guidelines on illumina.com for design.


Indexing PCR
  1. Add 2 uL of a unique RPi primer (10uM) in each library.
  2. Add 37.5 uL of PCR mix:
ReagentVolume (uL)
2X NEBNext High Fidelity mastermix25
RP1 primer 10uM2
ultra-pure water10.5
Total reaction volume is 50 uL

Incubate with the following program in a PCR machine:
98oC for 30 seconds
8-11 cycles of:
  • 98oC for 10 seconds
  • 60oC for 30 seconds
  • 72oC for 30 seconds
72oC for 10 minutes
Hold at 4oC

NOTES:
  • Each of the RPi primers (index primers) contains a unique index from the Illumina Truseq small RNA series (RPI series) and an overlapping sequence to the Illumina P7, introduced to the molecules during the previous RT step. Follow guidelines on illumina.com for design.
  • The RP1 primer (universal primer) contains an overlapping sequence to the Illumina P5, which is part of the DamID2 adapters sequence. Follow guidelines on illumina.com for design.
  • The exact number of cycles for the library PCR depends on the amount of input aRNA. We decide this empirically by comparing the height (FU) of the marker peak to the highest peak of the aRNA product distribution (excluding the adapter peak). For example, if the marker peak and the product are in similar FU levels, or if the product is much higher, we recommend 8 PCR cycles. Increase cycles accordingly for lower amounts.


Library purification
  1. Add 0.8 volume of undiluted beads. Incubate for 10 minutes.
  2. Put samples on a magnetic stand and incubate until all beads are bound to the magnet (liquid should look clear).
  3. Remove unbound liquid.
  4. Wash two times with freshly-made 80% ethanol.
  5. Air-dry beads until they look matte.
  6. Elute in 25 uL water. Leave for 10 minutes.
  7. Put on magnetic stand until clear and transfer eluate in a clean tube.

(REPEAT)
  1. Add 0.8 volume of undiluted beads. Incubate for 10 minutes.
  2. Put samples on a magnetic stand and incubate until all beads are bound to the magnet (liquid should look clear).
  3. Remove unbound liquid.
  4. Wash two times with freshly-made 80% ethanol.
  5. Air-dry beads until they look matte.
  6. Elute in 13 uL water. Leave for 10 minutes.
  7. Put on magnetic stand until clear and transfer eluate in a clean tube.


Library quantification and Sequencing
  1. Measure 1-2 uL of library with dsDNA Qubit assay to determine total library amount
  2. Run a max of 2ng of library in a Bioanalyzer (High Sensitivity DNA Assay) or equivalent to estimate size distribution
  3. Calculate library molarity based on Qubit concentration and size distribution
  4. Sequence with single-end or paired-end sequencing


Protocol references
Schmid, M., Durussel, T. & Laemmli, U. K. ChIC and ChEC; genomic mapping of chromatin proteins. Mol Cell 16, 147–57 (2004).

Skene, P. J. & Henikoff, S. An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites. Elife 6, (2017).

Ku, W. L. et al. Single-cell chromatin immunocleavage sequencing (scChIC-seq) to profile histone modification. Nat Methods 16, 323–325 (2019).

Zeller, P. et al. Single-cell sortChIC identifies hierarchical chromatin dynamics during hematopoiesis. Nat Genet 55, 333–345 (2023).

Gaza, H. V., Bhardwaj, V. & Zeller, P. Single-Cell Histone Modification Profiling with Cell Enrichment Using sortChIC. in Chromatin Immunoprecipitation (ed. Greulich, F.) vol. 2846 215–241 (Springer US, New York, NY, 2024).