CUT&RUN Chromatin Profiling of Human Kidney Tissue
Lynn Robbins, Jeannine M Basta, Michelle Pherson, Sabine Dietmann, Michael Rauchman
Abstract
Histone post-translational modifications are key epigenetic features that define gene regulatory elements in the genome. Identifying changes in the epigenome has the potential to uncover novel disease signatures. Chromatin immunoprecipitation with sequencing (ChIP-seq) is a commonly used method to define the chromatin landscape. A limitation of this method is that the amount of starting material (≥106 cells) does not lend itself to interrogation of kidney biopsies. Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a recently described method that provides genome-wide mapping of chromatin profiles for histone binding. In this method, cells are immobilized on beads, permeabilized with detergent (digitonin) and incubated with an antibody that recognizes specific histone post-translational modification in nucleosomes, DNA binding proteins (e.g., transcription factor) or other factors that associate with chromatin. After antibody binding, a fusion protein of Micrococcal nuclease and protein A/G (pAG-MNase) is added. The protein A/G moiety binds the antibody and the nuclease cuts accessible chromatin adjacent to the nucleosomes. The released DNA fragments, representing the genomic regions enriched for antibody binding, are column purified and used to construct a library for sequencing. Sequence is aligned to the human genome and peaks that represent statistically significant binding (e.g., histone H3 lysine 27 trimethylation, H3K27me3) are called using the Model-based Analysis for ChIP-seq Method (Macs2) that we have adapted for CUT& RUN.
Before start
Prepare 50mL Bead Activation Buffer, 10mL 100 mM CaCl2, and 10mL Stop Buffer without RNAse, glycogen, and spike-in DNA. Filter sterilize all and store at 4°C. These are good for 6 months. 1. The day before the experiment, prepare 3mL Wash Buffer/sample (sample= 1 CUT&RUN antibody reaction) and filter sterilize. Store at 4°C. This buffer is good for one week. Prepare 5% digitonin by dissolving 5 mg in 100µL DMSO. Store at 4°C. This buffer is good for one week.
*For human kidney cells we recover ~100,000 cells/mm3 of liquid nitrogen frozen tissue.
Steps
Antibody Buffer Prep and ConA Bead Activation
The day of the experiment, add protease inhibitors (PIs) to 1X concentration in 5mL of Wash Buffer and leave at Room temperature . Store the remaining Wash Buffer at 4°C for Day 2.
Make the Antibody Buffer (100µL/sample) by adding 2.0µL of 5% digitonin and 4.0µL 0.5 M EDTA to 1.0mL of Wash Buffer + PIs and put On ice. Leave the other 4mL at Room temperature.
Activate the Concanavalin A beads by transferring 11µL of beads/sample to a 1.5 ml low adhesion tube and place on magnet. Discard sup. Wash the beads 3x with 100µL cold Bead Activation Buffer/sample.
Resuspend the tube of beads in 11µL cold Bead activation buffer/sample. Aliquot 10µL beads/sample into new 1.5 mL low adhesion tubes and keep On ice until needed.
Tissue Prep and Binding Cells to Activated Beads
Thaw tissue On ice and measure size and weight. Transfer tissue to 1.5 mL low adhesion tube and add 100µL``Room temperature Wash Buffer + PIs/10mg tissue and dounce 10 times with a hand held pellet pestle.
Spin 0h 3m 0s at 600x g,0h 0m 0s at Room temperature and discard sup.
Add 100µL Wash Buffer + PIs/10mg tissue and pipet up and down 10 times with a 200 µl pipet.
Spin again and discard sup.
Add 500µL Wash Buffer + PIs and pipet up and down 10 times with a 200 µl pipet.
Wet a 30 µm filter with 500µL Wash Buffer + PIs and discard buffer from tube. Add tissue homogenate to the wet filter, and rinse the filter with an additional 500µL Wash Buffer + PIs.
Antibody Binding
Count cells using a hemocytometer and aliquot enough cells (50,000-500,000) per antibody reaction into each of the prepared tubes with activated ConA beads.
Gently vortex and let sit at Room temperature for 0h 10m 0s.
Place tubes on magnet and remove sup. Add 100µL cold Antibody Buffer to each tube and vortex gently. Add 1.0-2.0 µl antibody (negative control IgG or specific antibody) to each tube and vortex gently. Nutate overnight at 4°Cwith caps elevated.
Binding of pAG-MNase
In the morning, prepare 2.5mL Digitonin Buffer/sample by adding 5µL of 5% digitonin in DMSO to 2.5mLcold Wash Buffer. Add protease inhibitors to 1X. Keep Digitonin Buffer with PIs on ice.
Place tubes on the magnet, let beads clear from the reaction and remove the sup. Leaving the tubes on the magnet, gently pipet 300µL of cold Digitonin Buffer over the beads and then remove the sup.
Repeat static washes 2 more times for a total of 3 washes.
Resuspend each tube of beads with 50µL cold Digitonin Buffer and chill 0h 2m 0s on ice.
Add 2.5µL CUTANA pAG-MNase (20X stock) to each tube and pipet to mix.
Incubate the tubes atRoom temperature for 0h 10m 0s.
Place tubes on the magnet and immediately add 300µL cold Digitonin Buffer on top of the beads and pAG-MNase.
Remove sup and do 2 more static washes with the tubes on the magnet.
Resuspend beads in 50µL cold Digitonin Buffer and chill 0h 2m 0s On ice.
Chromatin Digestion and Release
Add 1µL 100 mM CaCl2to each tube and gently vortex briefly.
Immediately place back on ice. Nutate 2h 0m 0s at 4°C.
In the meantime, prepare 300µL Reaction Stop Buffer by mixing 0.75µL of 20 mg/mL RNase, 0.75µLof 20 mg/mL glycogen, and Epicypher E.coli spike-in DNA (optional, 150 pg/150,000 cells) into 300µL Stop Buffer.
Add 33µL Stop Buffer to each reaction and vortex gently.
Incubate 0h 10m 0s at 37°C in a water bath to release the chromatin and degrade the RNA.
Spin briefly to collect sup, place the tubes on the magnet, let clear, and transfer sups into a new tube.
Purify DNA
Clean up reactions using CUTANA DNA Purification kit according to their instructions.
Elute in 20µL and quantify DNA recovery using a Qubit fluorometer per manufacturer's instructions. Use 1-2 µl to measure concentration.
Library Prep
Using up to 5 ng purified CUT&RUN enriched DNA, prepare Illumina library using the NEBNext Ultra II Library Kit for Illumina per manufacture's instructions with the noted modifications:
End repair modification: 0h 30m 0s at 20°C, 0h 30m 0s at 50°C, to avoid melting smaller fragments when antibodies used are for transcription factors.
Universal adapter ligation modification: dilute adapter 1:25 if starting with > 2.5 ng; dilute adapter 1:50 for 1.25-2.5 ng; dilute adapter 1:100 if starting with < 1.25 ng.
DNA cleanup using 1.1x AMPure XP beads to sample volume for histone modification antibodies, or 1.35x beads for transcription factor antibodies.
PCR and primer indexing according to cycling parameters below.
CUT&RUN-specific PCR cycling parameters:
a. 0h 0m 45s @ 98°C- activation of hot-start Q5 polymerase
b. 0h 0m 15s @98°C- DNA melting
c. 0h 0m 13s@ 60°C- hybrid primer annealing and short extension (<700bp)
d. Repeat "step b-c" for a total of 15-18 cycles, depending on the amount of input DNA. If the amount of input was undetectable, use 18 cycles.
e. 0h 1m 0s @ 72°C- final extension
DNA cleanup using 1.1x AMPure beads to sample volume (e.g. 55µL beads, 50µL PCR reaction).
Elute DNA in 30µL 0.1x TE buffer and use 1µL to quantify the purified PCR product using the Qubit Fluorometer as per manufacturer's instructions. Typical yield of a PCR DNA library: ~500-750 ng (15-25 ng/µl in 30 µl).
Agilent Bioanalyzer System
For each sample, prepare 5µL at 1-5 ng/µl for loading on the Agilent Bioanalyzer.
For each purified PCR DNA library for Illumina sequencing, load 1µL of 5 ng/µl sample on Agilent High Sensitivity DNA Chip (Cat# 5067-4626) as per manufacturer's instructions.
Confirm that positive control antibodies enriched for predominantly mononucleosome fragments (~275 bp peak with nucleosomes + sequence adapters).
Sequencing
For Illumina libraries 0.8 pm is loaded and sequenced at 10-50 million paired end reads on a NovaSeq 6000 platform.
Bionformatic Analysis
Trim fastq files to remove adapters using Cutadapt.
Command Line Example: cutadapt -j 10 -m 10 -a AGATCGGAAGAG -A AGATCGGAAGAG -o R1.trim.fastq.gz -p R2.trim.fastq.gz R1.fastq.gz R2.fastq.gz
Align trimmed fastq files to genome using Bowtie2 end to end mode.
Command Line Example: bowtie2 --end-to-end --very-sensitive --no-mixed --no-discordant --phred33 -I 10 -X 700 -p 10 -x genome -1 R1.trim.fastq.gz -2 R2.trim.fastq.gz -S sample.sam
Use SAMtools view and sort commands to extract aligned reads from the resulting sam file and convert to a sorted bam file. Index the sorted bam file using SAMtools index.
Command Line Example:
a. samtools view -bS -F 0x04 sample.sam > aligned.bam
b. samtools sort aligned.bam -o aligned.sort.bam
c. samtools index aligned.sort.bam
Calculate sample normalization factor based on % of aligned E. coli reads (as described in Epicypher Cut&Run protocol).
a. Calculate percent of aligned E. coli reads in all uniquely aligned reads (ex: 100,000 E. coli reads in 5,000,000 uniquely aligned reads = 2%).
b. Calculate normalization factor to make E. coli spike in signal equal across all samples (ex: 1/2% = 0.5).
Generate scaled, binned bigWig file using Deeptools bamCoverage command with --scaleFactor parameter.
Command Line Example: bamCoverage -b aligned.sort.bam --scaleFactor 0.5 --binSize 50 --outFileFormat bigwig -o sample.bw
Call peaks using Macs2. Use broad peak calling option for histone modifications.
Command Line Example: macs2 callpeak -t aligned.sort.bam -c IgG.sort.bam -f BAM -g mm -n sample.name --broad --max-gap 1000 --min-length 160 --keep-dup all --fe-cutoff 2.7
