Chromatin Immunoprecipitation of Murine Brown Adipose Tissue

The animal handling steps of the protocol have been approved by Boston University’s Institutional Animal Care and Use Committee (IACUC).

1. Day 1: Dissection and Preparation of BAT for Chromatin Immunoprecipitation (ChIP)

1. Euthanize mice using a carbon dioxide (CO₂) chamber, and perform the dissection immediately afterwards. Spray mouse fur with 70% ethanol before incision. Place the mouse with its back facing up, then cut open the skin along the neck. Locate the BAT directly under the skin between the shoulders (interscapular; it appears as two lobes, butterfly-shaped, with a thin layer of white fat that needs to be carefully removed⁷).
   NOTE: For a 3 month-old C57BL/6J mouse that is fed a regular chow diet (mouse weight at this age ranges between 27 and 30 g), each BAT lobe is approximately 1 cm long. The total weight of the BAT depot is approximately 0.15 g in both male and female mice.
2. Use 1 BAT pad for 3 ChIPs. Multiple BAT pads can be simultaneously processed until sonication.
3. Crosslink each BAT pad in 10 mL of phosphate-buffered saline (PBS) containing 1% formaldehyde for 15 min rotating at 150 rpm at room temperature (RT).
4. Quench crosslinking by adding 2.5 M glycine to a final concentration of 125 mM for 5 min, rotating at 150 rpm at RT.
5. Transfer the BAT pad to 500 µL of hypotonic lysis buffer (10 mM HEPES, 1.5 mM MgCl₂, 10 mM KCl, 0.5 mM DTT, 0.2 mM PMSF) and add 2 stainless steel beads (size: 3.2 mm diameter, see Table of Materials) for each BAT pad. Use a bead-based tissue homogenizer (see Table of Materials) to shred the tissue.
6. Start with 5 min at max power. If needed, extend the time until the tissue is homogenized and single cells are separated. Transfer the samples into a new tube and centrifuge at 4 °C at 16,000 x g for 10 min.
7. After centrifugation, transfer the supernatant into a new tube and keep the cell pellet on ice. Centrifuge the supernatant at 4 °C at 16,000 x g for 10 min to prevent loss of floating cells. Discard the final supernatant and resuspend both cell pellets together in 300 µL of SDS lysis buffer (SDS 1%; EDTA 10 mM; Tris-HCl pH 8, 50 mM) with 1x protease inhibitor (see Table of Materials). Incubate on ice for 10 min.
8. Sonicate the lysates to generate DNA fragments 200–500 base pairs long. After sonication, remove debris by centrifugation for 10 min at 16,000 x g at 4 °C and check the success of sonication via DNA electrophoresis on a 1% agarose gel. For a medium-size gel, run at 120 V; a good separation is achieved after 45 min.
   NOTE: Sonication optimization may be required to achieve proper size of fragments. With the chosen sonicator (see Table of Materials), this requires 2 times at 320 W output power and 20 KHz frequency for 10 min, using cycles of 30 s on/30 s off.
9. Dilute the supernatant fraction 10-fold (final volume is 3 mL for each BAT pad) in ChIP dilution buffer (SDS 0.01%; Triton 1.1%; EDTA 1.2 mM; Tris-HCl pH 8, 16.7 mM; NaCl 167 mM) with 1x protease inhibitor. Keep aside 1% of this chromatin solution (equal to 30 µL for 3 mL) as the ChIP input. Keep the inputs overnight at 4 °C (until step 2.4).
   Note: The above fraction will provide the input reference for relative quantification of immunoprecipitated material compared to the amount of DNA present in each sample prior to immunoprecipitation.
10. Add the ChIP antibody (see Table of Materials for the antibodies used here as an example) to 1 mL of chromatin solution and incubate overnight at 4 °C with rotation. Perform parallel immunoprecipitation with corresponding pre-immune IgG, if available, or normal IgG of the same species (e.g., regular rabbit IgG if the antibody is produced in a rabbit). Alternatively, save 1 mL of chromatin solution for a no-antibody control.
    NOTE: The optimal amount of antibody should be determined experimentally for each new antibody, if the antibody concentration is not known. Otherwise, 2–5 µg of antibody is a reasonable starting amount to use. Use ChIP-grade antibodies whenever possible, or antibodies that have been validated for immunoprecipitation.

2. Day 2: Collection of the Immune Complexes

1. Add 50 µL of Protein A Agarose 50% slurry to the immune complexes and incubate for 1 h at 4 °C with rotation at 150 rpm.
2. Pellet the beads by centrifugation for 1 min at 100 x g at 4 °C. Perform sequential washes of the beads with buffers of increasing stringency.
   1. First, wash with low-salt wash buffer (150 mM NaCl; Tris-HCl pH 8, 20 mM; EDTA 2 mM; Triton-X 1%; SDS 0.1%), then with high-salt wash buffer (500 mM NaCl; Tris-HCl pH 8, 20 mM; EDTA 2 mM, Triton-X1%; SDS 0.1%), then with LiCl wash (0.25 M LiCl; Tris-HCl pH 8, 10 mM; EDTA 1 mM; Igepal 1%; deoxicholate 1%), and finally with 1x TE (Tris-HCl pH 8, 10 mM; EDTA).
   2. Perform all washes on 0.45 µm PVDF centrifugal filter columns (Table of Materials) by first transferring the beads in the columns using 500 µL of low-salt wash buffer. Pellet the beads in the columns for 3 min at 2,500 x g. Discard the flow-through and repeat 1 time for all the wash buffers listed in step 2.2.1.
3. After the washes are completed, elute the immune complexes by adding 300 µL of elution buffer 1% SDS, 0.1 M NaHCO₃) to the pelleted beads in the columns. Incubate at RT for 30 min on a shaker at 400 rpm. Collect the eluate by spinning down columns for 3 min at 2,500 x g in a fresh tube.
4. Reverse the crosslinks by incubating the eluate and inputs collected in step 1.9 at 65 °C overnight.

3. Day 3: Recovery of DNA with Phenol/Chloroform Extraction

1. Add 300 µL of phenol:chloroform:IAA, 1:1 to the eluate and inputs. Vortex for 10 s.
2. Spin at 4 °C for 15 min at 21,000 x g. Transfer the supernatant to a fresh tube. Add 3 µL of glycogen, 30 µL of 3 M sodium acetate, and 750 µL of cold 100% ethanol. Vortex for 10 s. Store at -80 °C for 2 h.
3. Spin down at 21,000 x g at 4 °C for 20 min, keep the pellet, and discard the supernatant. Wash pellet with 1 mL of cold 70% EtOH, then spin down at 21,000 x g at 4 °C for 5 min. Discard the supernatant and air-dry the pellet.
4. Resuspend the pellet in 70 µL of DNAse-free water.
5. Proceed to step 4 for testing chromatin occupancy on specific genomic regions by quantitative polymerase chain reaction (qPCR) or to step 5 to prepare samples for sequencing.

4. Analysis of ChIP using qPCR (Single/Multiple Genes Readout)

1. Dilute the input DNA from step 3.4 to generate a standard reference curve. Serial dilutions of 1/10, 1/100, and 1/1000 are usually sufficient to capture the linear range, but further dilutions may be required for more concentrated samples.
2. For each primer pair (here, we have used primers targeting NDUFV1 and TOMM20 promoters), prepare two sets of qPCR reactions: one using the diluted input samples from step 4.1, and a second with the DNA samples isolated by immunoprecipitation from step 3.4. Perform each reaction in triplicate.
   NOTE: Reactions can be set up in parallel for multiple genes using 96- or 384-well plates.
3. Set up a reaction volume of 10 μL per well with 5 μL of 2x PCR mix (Table of Materials), 1 μL of primer mix (1 μM forward primer and 1 μM reverse primer), and 4 μL of DNA from ChIP (or the input control).
4. Briefly spin down the plate.
5. Run the qPCR reactions on a real-time PCR system using the protocol recommended by the manufacturer for reagent detection (Table of Materials). Here we have used the following conditions: 1) 1s at 95°C for 1 cycle; 2) 1 s at 95 °C followed by 20 s at 60 °C for 45 cycles; 3) 15 s at 95 °C followed by 60 s at 60 °C followed by 15 s at 95 °C for 1 cycle.
   NOTE: Check the dissociation curve: the presence of one peak in the thermal dissociation plot suggests a single amplicon is generated from the PCR reaction. If more than one peak is visualized, this is potentially indicative of multiple, non-specific amplicons; in which case, we recommend the design of alternative primer pairs.
6. Determine the linear phase of exponential amplification of the PCR reaction for each primer set by calculating the standard curve using the series of diluted genomic DNA in step 4.1. according to the Ct (cycle threshold) value.
7. If the Ct value of DNA from ChIP and input control are within the linear range of Ct value, calculate the enrichment of DNA from ChIP relative to the input, according to the dilution factor of DNA from ChIP and input control in step 4.1.

5. Amplification of DNA from ChIP for High-throughput Sequencing (Genome-wide Readout)

NOTE: This step can be outsourced to an academic core facility or commercial sequencing company when sequencing capabilities are not available in-house.

1. Prepare a DNA library from the extracted DNA with an appropriate ChIP library preparation kit (see Table of Materials) following the manufacturer’s instructions.
2. Sequence the library on a high-throughput sequencing system, using 50 bp single-end as readout (see Table of Materials).
   NOTE: According to ChIP-seq guidelines suggested by ENCODE⁶, a minimum of 10 million reads is recommended for mammalian genomes.

6. Raw Data Analysis

1. Perform quality control on raw sequencing reads using FastQC⁸.
   NOTE: Common sequencing quality metrics include per base sequence quality, per base sequence content, per sequence GC content, sequence length, and sequence duplication levels. Generally, a read quality score of Q > 30 across each base position is indicative of high-quality sequencing results. The distribution of GC content across all reads should roughly resemble a normal distribution, with a peak centered around the species’ actual genomic GC content percentage.
2. Remove any sequencing adapter contamination and low-quality reads through the read trimming utility Trimmomatic⁹.
   NOTE: The implemented default parameters specify the removal of platform dependent adapters, removal of leading and trailing low-quality bases, and use of a 4-base wide sliding window to trim reads when the average quality per base drops below a specified threshold.
3. Align processed reads to the mouse MM10 reference genome¹⁰ with the Bowtie2 aligner¹¹ using default parameters.
4. Filter aligned reads by removing those with a Bowtie2 mapping quality score (MAPQ) of less than 10 using Samtools¹².
5. Determine various post-alignment quality metrics such as percent reads mapped, strand cross-correlation, cumulative read coverage, and sample replicate reproducibility­.
   Note: Various packages including SPP¹³, ChIPqc¹⁴, and Deeptools¹⁵ are available to calculate these quality metrics.
6. Perform peak calling analysis with an input control or appropriate KO sample using the MACS algorithm (MACS2)¹⁶ with default parameters.
7. Remove any called peaks that overlap with known blacklisted regions¹⁷ from the mm10 annotation using Bedtools¹⁸.
   NOTE: Blacklisted regions are areas in the genome that have been shown to have high signal or read counts independent of cell line or sequencing technique. These regions display artificially high artifact signals in certain regions of the genome that can be filtered out of functional genomic sequencing experiments. All replicates should be processed independently through the pipeline and may then be used for irreproducibility discovery rate (IDR). IDR is employed to measure the reproducibility of findings between replicates through the method as described by Li, Brown, Huang, and Bickel^19,20. The IDR method quantitatively assesses consistency between replicates and is recommended by ENCODE and modENCODE as part of their ChIP-seq guidelines.
8. Use statistically significant peaks for various downstream analyses such as gene ontology^21,22, enrichment, or motif analysis²³.