Multi-omics Techniques for Profiling Chromatin State Transitions in Low-input Primary Mouse Cholangiocytes

Summary

This manuscript presents a comprehensive protocol for low-input ChIP-seq to profile histone modifications and ATAC-seq to assess chromatin accessibility in small amounts of primary mouse cholangiocytes.

Abstract

Polycystic liver disease (PLD) is a hereditary disorder characterized by the formation of fluid-filled cysts derived from cholangiocytes, leading to progressive disease and a significant reduction in patients' quality of life. Current treatments for PLD are inadequate, emphasizing the need for novel therapeutic strategies. The role of epigenetic regulation in PLD progression, particularly chromatin accessibility and histone modifications, remains underexplored. Traditional epigenetic profiling techniques, such as ChIP-seq and DNase-seq, require large numbers of cells, which are difficult to obtain from primary cholangiocytes. To address this, we optimized low-input ChIP-seq and ATAC-seq protocols for low numbers of primary cholangiocytes. These approaches allow for the analysis of histone modifications and chromatin accessibility with minimal cell input. Low-input ChIP-seq utilizes micrococcal nuclease (MNase) for DNA fragmentation, while ATAC-seq employs Tn5 transposase to capture open chromatin regions. These multi-omics techniques provide valuable insights into chromatin state dynamics during cholangiocyte fate transitions in PLD and other biliary diseases. Importantly, the optimized protocols are confidently applicable to other low-input primary cells, enabling the exploration of epigenetic mechanisms across various cellular contexts. This work presents a systematic approach for studying chromatin state alterations, contributing to the development of epigenetic-based therapeutic strategies for PLD and related diseases.

Introduction

PLD is an inherited disorder characterized by the development of multiple fluid-filled cysts derived from cholangiocytes. As these cysts progressively expand, they severely impact patients' quality of life^1,2. Existing treatment strategies for PLD are inadequate, providing only limited benefits while frequently leading to high recurrence rates and complications^3,4. Therefore, there is a pressing need for safer and more effective therapeutic approaches to meet the unresolved clinical challenges in PLD treatment.

Under normal conditions, cholangiocytes remain quiescent, whereas in PLD, they exhibit excessive proliferation, a key driver of disease progression^5,6. The molecular mechanisms underlying this cystic transition remain unclear. While epigenetic regulation, including chromatin accessibility and histone modifications, plays a vital role in cell fate transitions^7,8, its role in PLD progression remains understudied. However, many epigenetic profiling techniques require a large number of cells. Traditional ChIP-seq, which relies on chromatin fragmentation by sonication, as well as chromatin accessibility assays such as DNase-seq and MNase-seq, typically require over 10⁶ cells-far exceeding the number obtainable from primary cholangiocytes.

This manuscript provides a comprehensive protocol for low-input ChIP-seq^9,10,11 to profile histone modifications and ATAC-seq^11,12,13 to assess chromatin accessibility in low numbers of primary cholangiocytes. Low-input ChIP-seq employs MNase to fragment DNA⁹, while ATAC-seq captures DNA fragments from open chromatin regions using Tn5 transposase¹². Multi-omics analysis utilizing these approaches offers valuable insights into chromatin state dynamics during cholangiocyte fate transitions in PLD and other biliary diseases, thereby facilitating the development of epigenetic-targeted therapies.

Protocol

1. Preparation of solutions and cholangiocytes for ATAC-seq

1. At least 1 day prior to the ATAC-seq experiment, prepare the ATAC Lysis Buffer using the reagents listed in Table 1.
   NOTE: ATAC Lysis Buffer can be stored at 4 °C in the dark for at least 1 month.
2. For freshly isolated primary cholangiocytes¹⁴, count the cells using a hemocytometer immediately after isolation and proceed with the ATAC-seq experiment.
   NOTE: The sooner the better to minimize the impact on cell viability. However, if kept on ice, proceeding within 2 h is generally not a problem.

2. ATAC-seq

NOTE: Use 100,000 cholangiocytes for ATAC-seq. Keep the samples and solutions on ice. For the ATAC-seq experiment, the entire procedure takes approximately 2 days.

1. Centrifuge at 500 × g for 5 min at 4 °C.
2. Discard the supernatant, add 200 µL of ATAC Lysis Buffer containing Protease Inhibitor, gently pipette to mix, and incubate on ice for 20 min. For the first 10 min, gently pipette to mix.
3. Centrifuge at 1,500 × g for 10 min at 4 °C.
4. Prepare the Transposition Reaction Buffer (50 µL) by mixing 3 µL of Enzyme Mix from the ATAC DNA Library Prep Kit, 10 µL of 5x Buffer from the ATAC DNA Library Prep Kit, and 37 µL of Nuclease-free water. Mix thoroughly.
5. Carefully discard the supernatant, place the collected nuclear pellet on ice, and immediately add 50 µL of Transposition Reaction Buffer. Gently resuspend and incubate at 37 °C for 60 min, flicking the tube bottom every 10 min to mix.
   NOTE: Prepare the Transposition Reaction Buffer before discarding the supernatant to prevent the cells from drying out.
6. Purify DNA using the PCR Purification Kit according to the manufacturer's instructions.
   NOTE: Residual Protein G beads from the cholangiocyte isolation process¹⁴ could be removed in this step. DNA samples can be stored at -80 °C for long-term preservation.
7. Perform library construction using the ATAC DNA Library Prep Kit according to the manufacturer's instructions.
   NOTE: Keep all samples and solutions on ice unless otherwise specified. While procedures may differ among other kit brands, all are compatible with our protocol.
   1. PCR Enrichment: Prepare the PCR Enrichment Mix (50 µL) in a PCR tube by mixing the following components in the specified order: 10 µL of DNA sample (from step 2.6), 14 µL of Nuclease-free water, 10 µL of 5x TAB, 5 µL of PPM, 1 µL of TAE, 5 µL of P5 primer, and 5 µL of P7 primer. Gently pipette to mix.
   2. Transfer the tube to a PCR instrument and run the following program: (72 °C for 3 min) x 1 cycle, (98 °C for 30 s) x 1 cycle, (98 °C for 15 s, 60 °C for 30 s, 72 °C for 3 min) x 15 cycles, and (72 °C for 5 min) x 1 cycle, and hold at 4 °C.
   3. Purification: Bring the DNA Clean beads to room temperature for 30 min before use, then perform a two-step magnetic bead purification.
      1. First round: Add 30 µL of DNA Clean beads to the PCR products (from step 2.7.2), gently pipette to mix, and incubate at room temperature for 5 min. Place the PCR tube on a magnetic separator and wait until the solution becomes clear (~5 min). Carefully transfer the supernatant to a new tube and discard the DNA Clean beads.
      2. Second round: Add 7.5 µL of DNA Clean beads to the collected supernatant (from step 2.7.3.1), gently pipette to mix, and incubate at room temperature for 5 min. Place the tube on a magnetic separator and wait until the solution becomes clear (~5 min). Carefully discard the supernatant.
   4. Keep the tube on the magnetic separator and rinse the DNA Clean beads for 2 x 30 s with 200 µL of freshly prepared 80% ethanol at room temperature. Carefully discard the supernatant after each rinse. With the tube still on the magnetic separator, open the tube lid and air-dry the beads for 10 min. Finally, add 22 µL of Nuclease-free water to elute the DNA.
      NOTE: DNA samples can be stored at -80 °C for long-term preservation.
8. Sequence the libraries using a standard next-generation sequencer.
   NOTE: We recommend obtaining at least 50 million pair-end reads.

3. Preparation of solutions and cholangiocytes for Low-input ChIP-seq

1. At least 1 day prior to the low-input ChIP-seq experiment, prepare the ChIP Lysis Buffer, ChIP MNase Buffer, ChIP Stop Buffer, ChIP 2x RIPA Buffer, ChIP RIPA Buffer, and ChIP LiCl Buffer using the reagents listed in Table 2, Table 3, Table 4, and Table 5.
   NOTE: The above solutions can be stored at 4 °C for at least 1 month.
2. For freshly isolated primary cholangiocytes¹⁴, count the cells using a hemocytometer immediately after isolation and proceed with the low-input ChIP-seq experiment.
   NOTE: The sooner the better to minimize the impact on cell viability. However, if kept on ice, proceeding within 2 h is generally not a problem.

4. Day 1 of Low-input ChIP-seq: Cell lysis, MNase digestion, and Antibody incubation

NOTE: For the low-input ChIP-seq experiment, the entire procedure takes approximately 3 days.

1. Divide cholangiocytes into aliquots of 100,000 cells per tube and keep the samples and solutions on ice.
   NOTE: If performing a low-input ChIP-seq experiment for multiple histone modifications, it is acceptable to scale up the volume proportionally before adding the histone modification antibody. The reagent volumes in the following steps are based on one tube containing 1 × 10⁵ cells.
2. Centrifuge at 2,000 × g for 5 min at 4 °C.
3. Discard the supernatant, add 19 µL of ChIP Lysis Buffer containing Protease Inhibitor, gently pipette to mix, and incubate on ice for 10 min. Avoid generating bubbles.
4. Add 19 µL of ChIP MNase Buffer and gently pipette to mix.
5. Add 2 µL of MNase (0.01 U/µL) and gently pipette to mix. Incubate at 37 °C for 5 min.
   NOTE: The incubation time is critical. The final concentration of MNase is 0.0005 U/µL. The optimal concentration of MNase can be determined based on cell type and number.
6. Immediately place the sample on ice, add 5 µL of ChIP STOP Buffer to terminate digestion, and gently pipette to mix.
7. Add 45 µL of ChIP 2x RIPA Buffer containing Protease Inhibitor, gently pipette to mix, and further incubate on ice for 10 min.
8. Centrifuge at 20,000 × g for 15 min at 4 °C. Slowly transfer the supernatant to a new tube.
   NOTE: Residual Protein G beads from the cholangiocyte isolation process¹⁴ could be removed in this step.
9. Add 40 µL of ChIP RIPA Buffer.
10. For Immunoprecipitation (IP) samples, add 1 µg of antibody per sample and incubate the suspension on a rotator (20 rpm) at 4 °C overnight.
11. For Input samples, do not add any antibody and incubate the suspension on a rotator (20 rpm) at 4 °C overnight.

5. Day 2 of Low-input ChIP-seq: Immunoprecipitation, Wash and Elution

NOTE: For IP samples, follow steps 5.1-5.7. For Input samples, follow steps 5.8-5.12.

1. Prewash Protein G beads with 1 mL of ice-cold ChIP RIPA Buffer.
2. Add 400 µg of Protein G beads to the samples and incubate with rotation (20 rpm) at 4 °C for 2 h.
3. Wash the beads for 5 x 5 min with 150 µL of ice-cold ChIP RIPA Buffer for 5 min at 4 °C on a rotator (20 rpm). Remove the supernatant using a magnetic separator.
4. Wash the beads with 150 µL of ice-cold ChIP LiCl Buffer for 5 min at 4 °C on a rotator (20 rpm). Remove the supernatant using a magnetic separator.
5. Add 30 µL of ChIP Elution Buffer (29 µL of Nuclease-free water and 1 µL of Proteinase K) to resuspend the beads and shake at 900 rpm at 55 °C for 90 min. Gently flick the tube periodically to keep the beads in suspension.
6. Collect the supernatant using a magnetic separator and slowly transfer it to a new tube. Incubate at 72 °C for 40 min to inactivate Proteinase K.
7. Measure DNA concentration using a fluorometer.
   NOTE: DNA samples can be stored at -80 °C for long-term preservation.
8. For Input samples, add 1 µL of Proteinase K and shake at 900 rpm at 55 °C for 90 min.
9. Purify DNA using phenol-chloroform extraction. Add 130 µL of DNA Extraction Reagent and vortex vigorously until the solution turns pink. Centrifuge at 20,000 × g for 5 min.
10. Transfer the supernatant to a new tube, add 130 µL of ice-cold isopropanol, and then add 1 µL of glycogen and 6.5 µL 3M NaAc. Centrifuge at 20,000 × g for 15 min.
11. Discard the supernatant, wash once with 1 mL of 70% ethanol, and centrifuge at 20,000 × g for 15 min. Dry the pellet and resuspend it in 30 µL of Nuclease-free water.
12. Measure DNA concentration using a fluorometer.
    NOTE: DNA samples can be stored at -80 °C for long-term preservation.

6. Day 3 of low-input ChIP-seq: Library construction and Sequencing

1. Perform library construction using a ChIP DNA Library Prep Kit according to the manufacturer's instructions.
   NOTE: The steps include End Preparation, Adapter Ligation, and Library Amplification. Pipette 5 ng of ChIP samples (Input and IP samples) for library construction. Keep all samples and solutions on ice unless otherwise specified. While procedures may differ among other kit brands, all are compatible with our protocol.
2. End Preparation: Prepare the End Preparation Reaction Buffer (65 µL) in a PCR tube by mixing 5 ng of DNA sample (from step 5.6 or step 5.11), 15 µL of End Prep Mix 4, 6.5 µL of Tris EDTA, and Nuclease-free water to a final volume of 65 µL. Gently pipette to mix. Transfer the tube to a PCR instrument and run the following program: 20 °C for 15 min, 65 °C for 15 min, and hold at 4 °C (Hot Lid: 105 °C).
3. Adapter Ligation: Dilute DNA Adapter S at a ratio of 1:100. Prepare the Adapter Ligation Reaction Buffer (100 µL) in a PCR tube by mixing 65 µL of End Preparation Products (from step 6.2), 25 µL of Rapid Ligation buffer 2, 5 µL of Rapid DNA ligase, and 5 µL of diluted DNA Adapter S. Gently pipette to mix. Transfer the tube to a PCR instrument and run the following program: 20 °C for 15 min, followed by a hold at 4 °C (Hot Lid: 105 °C).
4. Purification:
   1. Bring the DNA Clean beads to room temperature for 30 min before use. Add 60 µL of DNA Clean beads to the Adapter Ligation products (from step 6.3). Gently pipette to mix and incubate at room temperature for 5 min.
   2. Place the PCR tube on a magnetic separator and wait until the solution becomes clear (~5 min). Carefully discard the supernatant.
   3. Keep the tube on the magnetic separator. Rinse the DNA Clean beads for 2 x 30 s with 200 µL of freshly prepared 80% ethanol at room temperature. Carefully discard the supernatant after each rinse.
   4. With the tube still on the magnetic separator, open the tube lid and air-dry the beads for 5 min. Finally, add 22.5 µL of 10 mM Tris-HCL (PH 8.0) to elute the DNA.
5. Library Amplification: Prepare the Library Amplification Reaction Buffer (50 µL) in a PCR tube by mixing 20 µL of Purification products (from step 6.4), 2.5 µL of i5 PCR Primer, 2.5 µL of i7 PCR Primer, and 25 µL of Amplification Mix. Gently pipette to mix. Transfer the tube to a PCR instrument and run the following program: (95 °C for 3 min) x 1 cycle, (98 °C for 20 s, 60 °C for 15 s, 72 °C for 30 s) x 18 cycles, (72 °C for 5 min) x 1 cycle, and hold at 4 °C.
6. Purification: Bring the DNA Clean beads to room temperature for 30 min before use, then perform a two-step magnetic bead purification.
   1. First round: Add 30 µL of DNA Clean beads to the Library Amplification products (from step 6.5). Gently pipette to mix and incubate at room temperature for 5 min. Place the PCR tube on a magnetic separator and wait until the solution becomes clear (~5 min). Carefully transfer the supernatant to a new tube and discard the DNA Clean beads.
   2. Second round: Add 7.5 µL of DNA Clean beads to the collected supernatant. Gently pipette to mix and incubate at room temperature for 5 min. Place the tube on a magnetic separator and wait until the solution becomes clear (~5 min). Carefully discard the supernatant. Keep the tube on the magnetic separator. Rinse the DNA Clean beads for 2 x 30 s with 200 µL of freshly prepared 80% ethanol at room temperature. Carefully discard the supernatant after each rinse. With the tube still on the magnetic separator, open the tube lid and air-dry the beads for 10 min. Finally, add 22.5 µL of 10 mM Tris-HCl (PH 8.0) to elute the DNA.
      NOTE: DNA samples can be stored at -80 °C for long-term preservation.
7. Sequence the libraries in a standard next-generation sequencer.
   NOTE: We recommend obtaining at least 10-20 million pair-end reads.

7. Data analysis of ATAC-seq and Low-input ChIP-seq

1. Preparing the analysis virtual environment (miniconda)
   1. Download Miniconda3 from the website using the following commands and follow the installation instructions of Miniconda.
      wget https://mirrors.tuna.tsinghua.edu.cn/anaconda/miniconda/Miniconda3-4.7.12.1-Linux-x86_64.sh
      bash Miniconda3-4.7.12.1-Linux-x86_64.sh
   2. Create a virtrual environment for analysis pipeline of both ATAC-seq and ChIP-seq.
      conda create -n NGSpipeline
      conda activate NGSpipeline
      conda install bioconda::fastqc bioconda::multiqc bioconda::trim-galore bioconda::bowtie2 bioconda::samtools bioconda::macs2 bioconda::deeptools bioconda::picard bioconda::igv
   3. Download the reference genome sequence file and make an alignment index for bowtie2 alignment.
      wget https://ftp.ebi.ac.uk/pub/databases/gencode/Gencode_mouse/release_M25/GRCm38.p6.genome.fa.gz
      gunzip GRCm38.p6.genome.fa.gz
      bowtie2-build-s GRCm38.p6.genome.fa GRCm38_bowtie2_index
2. Perform quality control of ATAC-seq reads and ChIP-seq reads with FastQC and summarize the quality control report using MultiQC to ensure the GC bias of the sequencing results and enough library complexity for downstream analysis. Discard sequence library with significant different quality control results.
   fastqc -o <output directory><seqfile1> <seqfile2> …… <seqfileN>
   multiqc <output directory>
3. Trim sequencing adaptor using trim galore.
   trim_galore -q 20 --phred33 --stringency 3 --length 25 -e 0.1 --paired -j <thread number> -o <output directory> <paired-end reads 1> <paired-end reads 2>
4. Map trimmed reads to mm10 reference genome assembly with Bowtie2 using default parameters, and pass addition parameters (--very-sensitive -X 2000) to bowtie2 for ATAC-seq due to wide length distribution of ATAC-seq fragment.
   1. For ATAC-seq:
      bowtie2 -p <thread number> --very-sensitive -X 2000 -x <genome index from step 7.1.3> -1 <cleaned fastq file from 4.2 read 1> -2 <cleaned fastq file from 4.2 read 2> | samtools view -S -O BAM -@ <thread number> -o <output bam file>
   2. For ChIP-seq:
      bowtie2 -p <thread number> -x <genome index from step 7.1.3> -1 <cleaned fastq file from 4.2 read 1> -2 <cleaned fastq file from 4.2 read 2> | samtools view -S -O BAM -@ <thread number> -o <output bam file>
5. Remove reads with low alignment score (q30) and reads aligned to mitochondria genome and non-standard chromosome.
   samtools view -bh -q 30 -f 2 <input bam file> | samtools view -h | grep -v 'chrM' | grep -v 'random' | grep -v 'chrUn' | samtools sort -l 9 -O BAM -o <output filtered bam file>
6. Use Picard MarkDuplicates command to remove PCR duplicates reads.
   picard MarkDuplicates I=<input filtered bam file> O=<output remove duplication bam file> M=<remove duplication report file> REMOVE_DUPLICATES=true
7. Call peak using MACS2 callpeak command with parameter -f BAMPE -g mm -B -q 0.01.
   macs2 callpeak -f BAMPE -t <remove duplication bam file > -g mm -n <output prefix> -B -q 0.01
8. Generate BigWig files using Deeptools BamCoverage command.
   samtools index <remove duplication bam file> -@ 4
   bamCoverage -b <remove duplication bam file> -o <output BigWig file> -bs 50 -p <thread number> --effectiveGenomeSize2652783500--normalizeUsing RPGC -e -ignoreDuplicates
9. Use IGV genome browser to view the narrowPeak file from macs2 and BigWig files from Deeptools BamCoverage.

Results

To generate the chromatin landscape of primary cholangiocytes, we optimized the low-input ChIP-seq and ATAC-seq protocols for low numbers (~100,000) of primary cholangiocytes. Agarose gel electrophoresis results for primary cholangiocytes indicated that for 1 × 10⁵ primary cholangiocytes, 0.02 U MNase at 37 °C for 5 min resulted in the production of mononucleosomes, which was identified as the optimal concentration (Figure 1).

Overview of ATAC-s...

Discussion

To systematically and comprehensively map the chromatin state dynamics underlying the state transition of cystic cholangiocytes, we successfully optimized low-input ChIP-seq and ATAC-seq for a limited number of primary cholangiocytes. Although this study focused on primary cholangiocytes, we are confident that the protocol can also be applied to other high-viability primary cells with limited availability. Similarly, while this study only presents the ChIP-seq analysis results of H3K9ac and H3K9me3, the protocol is equal...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5 M EDTA	Solarbio	E1170
1 M Tris-HCl (pH=7.5)	Solarbio	T1140
1 M Tris-HCl (pH=8.0)	Solarbio	T1150
3 M NaAc	Beyotime	ST342
8 M LiCl	Sigma	L7026
Agarose gel	Biosharp	BS081
ATAC DNA Library Prep Kit	Vazyme	TD501
CaCl2	Sangon Biotech	A501330	1 M stock
ChIP DNA Library Prep Kit	Vazyme	ND607
DNA Clean beads	Vazyme	N411
DNA Extraction Reagent	Solarbio	P1012
EGTA	Solarbio	E8050	100 mM (pH = 8) stock
Fluorometer	Invitrogen	Q33226
Glycogen	Thermo Scientific	R0561
Hemocytometer	QIUJING	XB.K.25.
Igepal CA-630	Sigma	I8896	10% stock
Magnetic separator	Promega	Z5342
MgCl2	Sangon Biotech	A100288	1.5 M stock
MNase	Sigma	N3755	0.01 U/µL stock
NaCl	Sangon Biotech	A610476	5 M stock
NP40	Solarbio	N8030
Nuclease-free water	Life Technologies	AM9937
PCR instrument	Applied Biosystems	4484073
PCR Purification Kit	QIAGEN	28106
Protease Inhibitor	Roche	04693132001
Protein G beads	Invitrogen	10004D
Proteinase K	TransGen	GE201-01
Rotator	Kylin-Bell	QB-528
SDS	Solarbio	S8010	10%  stock
Sodium deoxycholate	Sigma	S1827
Thermomixer comfort	Eppendorf	5355
Triton X-100	Solarbio	T8200
Tween-20	Solarbio	T8220
Software	Citation (PMID)/Company	Version	Website
Bowtie2	22388286	2.3.5.1	https://github.com/BenLangmead/bowtie2
Deeptools	27079975	3.4.3	https://deeptools.readthedocs.io/en/latest/
FastQC		0.12.1	https://www.bioinformatics.babraham.ac.uk/projects/fastqc/
IGV	21221095	2.12.3	https://igv.org/
MACS2	18798982	2.2.7.1	https://hbctraining.github.io/Intro-to-ChIPseq/lessons/05_peak_calling_macs.html
Miniconda	Anaconda	4.7.12.1	https://www.anaconda.com/
MultiQC	27312411	1.23	https://seqera.io/multiqc/
Picard		2.27.5	https://broadinstitute.github.io/picard/
samtools	33590861	1.6	https://www.htslib.org/
trim-galore		0.6.6	https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/