TITLE: CUT&amp;RUN Chromatin Profiling of Human Kidney Tissue
AUTHORS: Lynn  Robbins, Jeannine M Basta, Michelle Pherson, Sabine   Dietmann, Michael   Rauchman

[DESCRIPTION]
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 50 mL Bead Activation Buffer, 10 mL 100 mM CaCl2, and 10 mL Stop Buffer without RNAse, glycogen, and spike-in DNA. Filter sterilize all and store at 4 °C. These are good for 6 months. 
The day before the experiment, prepare 3 mL 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 100 µL 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. 





[GUIDELINES]
The full CUT&RUN protocol is from Epicypher (https://www.epicypher.com/content/documents/protocols/cutana-cut&run-protocol.pdf). Please reference this protocol for full details and required positive and negative controls for experiments. 

We outline a brief protocol with modifications for liquid nitrogen frozen human kidney tissue using 1.5 mL tubes and as little as 50,000 cells/CUT&RUN antibody reaction. Depending on the abundance of the protein target and specificity of the antibody, it is possible to use less than 50,000 cells; however, we recommend using 50,000-500,000 cells/reaction.  


[STEPS]
SECTION: Antibody Buffer Prep and ConA Bead Activation
1. The day of the experiment, add protease inhibitors (PIs) to 1X concentration in 5 mL of Wash Buffer and leave at Room temperature  .  Store the remaining Wash Buffer at 4 °C for Day 2.

SECTION: Antibody Buffer Prep and ConA Bead Activation
2. Make the Antibody Buffer (100 µL/sample) by adding 2.0 µL of 5% digitonin and 4.0 µL 0.5 M EDTA to 1.0 mL of Wash Buffer + PIs and put on ice. Leave the other 4 mL at Room temperature. 

 

SECTION: Antibody Buffer Prep and ConA Bead Activation
3. Activate the Concanavalin A beads by transferring 11 µL of beads/sample to a 1.5 ml low adhesion tube and place on magnet.  Discard sup.  Wash the beads 3x with 100 µL cold Bead Activation Buffer/sample.


 


SECTION: Antibody Buffer Prep and ConA Bead Activation
4. Resuspend the tube of beads in 11 µL cold Bead activation buffer/sample.  Aliquot 10 µL beads/sample into new 1.5 mL low adhesion tubes and keep on ice until needed.



SECTION: Tissue Prep and Binding Cells to Activated Beads
5. Thaw tissue on ice and measure size and weight. Transfer tissue to 1.5 mL low adhesion tube and add 100 µLRoom temperature Wash Buffer + PIs/10 mg tissue and dounce 10 times with a hand held pellet pestle. 

SECTION: Tissue Prep and Binding Cells to Activated Beads
6. Spin 3 min at 600 x g at Room temperature and discard sup. 

SECTION: Tissue Prep and Binding Cells to Activated Beads
7. Add 100 µL Wash Buffer + PIs/10 mg tissue and pipet up and down 10 times with a 200 µl pipet. 

SECTION: Tissue Prep and Binding Cells to Activated Beads
8. Spin again and discard sup. 
SECTION: Tissue Prep and Binding Cells to Activated Beads
9. Add 500 µL Wash Buffer + PIs and pipet up and down 10 times with a 200 µl pipet. 

SECTION: Tissue Prep and Binding Cells to Activated Beads
10. Wet a 30 µm filter with 500 µL Wash Buffer + PIs and discard buffer from tube. Add tissue homogenate to the wet filter, and rinse the filter with an additional 500 µL Wash Buffer + PIs. 

SECTION: Antibody Binding
11. 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. 
SECTION: Antibody Binding
12. Gently vortex and let sit at Room temperature  for 10 min. 

SECTION: Antibody Binding
13. Place tubes on magnet and remove sup. Add 100 µL 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. 




SECTION: Binding of pAG-MNase
14. In the morning, prepare 2.5 mL Digitonin Buffer/sample by adding 5 µL of 5% digitonin in DMSO to 2.5 mLcold Wash Buffer. Add protease inhibitors to 1X. Keep Digitonin Buffer with PIs on ice. 

SECTION: Binding of pAG-MNase
15. Place tubes on the magnet, let beads clear from the reaction and remove the sup. Leaving the tubes on the magnet, gently pipet 300 µL of cold Digitonin Buffer over the beads and then remove the sup. 




SECTION: Binding of pAG-MNase
16. Repeat static washes 2 more times for a total of 3 washes. 
SECTION: Binding of pAG-MNase
17. Resuspend each tube of beads with 50 µL cold Digitonin Buffer and chill 2 min on ice. 

SECTION: Binding of pAG-MNase
18. Add 2.5 µL CUTANA pAG-MNase (20X stock) to each tube and pipet to mix. 

SECTION: Binding of pAG-MNase
19. Incubate the tubes atRoom temperature for 10 min. 

SECTION: Binding of pAG-MNase
20. Place tubes on the magnet and immediately add 300 µL cold Digitonin Buffer on top of the beads and pAG-MNase. 

SECTION: Binding of pAG-MNase
21. Remove sup and do 2 more static washes with the tubes on the magnet. 
SECTION: Binding of pAG-MNase
22. Resuspend beads in 50 µL cold Digitonin Buffer and chill 2 min on ice. 

SECTION: Chromatin Digestion and Release
23. Add 1 µL 100 mM CaCl2 to each tube and gently vortex briefly. 

SECTION: Chromatin Digestion and Release
24. Immediately place back on ice. Nutate 120 min at 4 °C.

SECTION: Chromatin Digestion and Release
25. In the meantime, prepare 300 µL Reaction Stop Buffer by mixing 0.75 µL of 20 mg/mL RNase, 0.75 µLof 20 mg/mL glycogen, and Epicypher E.coli spike-in DNA (optional, 150 pg/150,000 cells) into 300 µL Stop Buffer. 

 

SECTION: Chromatin Digestion and Release
26. Add 33 µL Stop Buffer to each reaction and vortex gently. 

SECTION: Chromatin Digestion and Release
27. Incubate 10 min at 37 °C in a water bath to release the chromatin and degrade the RNA. 

SECTION: Chromatin Digestion and Release
28. Spin briefly to collect sup, place the tubes on the magnet, let clear, and transfer sups into a new tube. 
SECTION: Purify DNA
29. Clean up reactions using CUTANA DNA Purification kit according to their instructions. 
SECTION: Purify DNA
30. Elute in 20 µL and quantify DNA recovery using a Qubit fluorometer per manufacturer's instructions. Use 1-2 µl to measure concentration. 

 

SECTION: Library Prep
31. 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:
SECTION: Library Prep
31.1. End repair modification: 30 min at 20 °C, 30 min at 50 °C, to avoid melting smaller fragments when antibodies used are for transcription factors. 

SECTION: Library Prep
31.2. 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. 
SECTION: Library Prep
31.3. DNA cleanup using 1.1x AMPure XP beads to sample volume for histone modification antibodies, or 1.35x beads for transcription factor antibodies.
SECTION: Library Prep
31.5. CUT&RUN-specific PCR cycling parameters:
a. 45 s @ 98 °C- activation of hot-start Q5 polymerase
b. 15 s @98 °C- DNA melting
c. 13 s@ 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. 1 min @ 72 °C- final extension

SECTION: Library Prep
31.6. DNA cleanup using 1.1x AMPure beads to sample volume (e.g. 55 µL beads, 50 µL PCR reaction). 

SECTION: Library Prep
31.7. Elute DNA in 30 µL 0.1x TE buffer and use 1 µL 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). 

SECTION: Agilent Bioanalyzer System
32. For each sample, prepare 5 µL at 1-5 ng/µl for loading on the Agilent Bioanalyzer. 

 

SECTION: Agilent Bioanalyzer System
33. For each purified PCR DNA library for Illumina sequencing, load 1 µL of 5 ng/µl sample on Agilent High Sensitivity DNA Chip (Cat# 5067-4626) as per manufacturer's instructions.

 

SECTION: Agilent Bioanalyzer System
34. Confirm that positive control antibodies enriched for predominantly mononucleosome fragments (~275 bp peak with nucleosomes + sequence adapters).
SECTION: Library Prep
31.4. PCR and primer indexing according to cycling parameters below.
SECTION: Sequencing
35. For Illumina libraries 0.8 pm is loaded and sequenced at 10-50 million paired end reads on a NovaSeq 6000 platform. 
SECTION: Bionformatic Analysis
36. 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

 

SECTION: Bionformatic Analysis
37. 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

 

SECTION: Bionformatic Analysis
38. 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

 

SECTION: Bionformatic Analysis
39. 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).
SECTION: Bionformatic Analysis
40. 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

 

SECTION: Bionformatic Analysis
41. 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