ATAC-seq is a powerful technique to understand gene expression regulatory mechanisms. However, adipose tissue is difficult using this technique because of its large lipid contents, high mitochondrial contamination, and cellular heterogeneity. In this protocol, adipocyte-specific ATAC-sequencing was performed using fluorescence-activated nucleus sorting, which generates high-quality data with minimal mitochondrial DNA contamination.
This method can also be used in other tissues where cell-type-specific Cre mouse lines are available with nuclear labeling transgenic reporter lines. Begin the nucleus isolation by chilling the glass Dounce homogenizers on ice with one glass Dounce per sample. Then, add 7 milliliters of the NPB mix to each glass Dounce.
Then, place 50 to 100 milligrams of frozen adipose tissue in the glass Dounce, and cut it into a few smaller pieces using a long pair of scissors. Stroke 10 times with a loose pestle for all the samples, and then 10 times with a tight pestle. Filter the content through a 100 micrometre strainer into a new 50 milliliter tube before transferring the filtered homogenates to a 15 milliliter tube for centrifugation.
After removing the supernatant, resuspend the pellet in 500 microliters of PBS-N by gentle but thorough finger-tapping. Next, filter the suspension through a 40 micrometer strainer into a 50 milliliter tube and rinse the strainer with 250 microliters of PBS-N. Transfer the filtered nuclear suspension to a fluorescence-activated cell sorting or FACS tube using a micropipette.
Prepare the collection tube by adding 500 microliters of PBS-N to the new 1.5 milliliter tube, and invert it a few times to wet all inner surfaces with the buffer. Briefly spin the tubes to bring down all the liquid before placing them on ice until sorting. For FACS, use the FSC-A/FSC-H gating for singlets and FSC-A/SSC-A for small or large debris removal.
Gate for the mCherry/GFP-positive adipocyte nucleus population, and collect 10, 000 nuclei in each collection tube prepared earlier. For post-sorting nucleus preparation, add 500 microliters of PBS-N to each collection tube and mix it by inverting the tube a few times before centrifuging the collection tubes at 200 G for 10 minutes at 4 degrees Celsius. Completely remove the supernatant.
To prepare 25 microliters of Tn5 master mixture, mix 12.5 microliters of 2x tagmentation DNA buffer or TD buffer, 1.25 microliters of Tn5 transposase or TDE I enzyme, and 11.25 microliters of nuclease-free water. Next, add 25 microliters of Tn5 master mixture to each nucleus pellet and resuspend it by gentle pipetting. Incubate at 600 RPM for 30 minutes at 37 degrees Celsius using a thermal mixer.
Add 25 microliters of nuclease-free water to the 25 microliters of the mixture of Tn5 nucleus resuspension making the final volume of 50 microliters. Then, add 250 microliters of buffer PB and 5 microliters of 3 M sodium acetate with pH 5.2. After transferring the mixture to a column provided with the referenced kit, centrifuge it at 17, 900 G for 1 minute at room temperature.
After discarding the flowthrough, place a spin column back in the same collection tube. Add 750 microliters of buffer PE containing absolute ethanol to the tube. Centrifuge the column, discard the flowthrough, and place the spin column back into the same collection tube.
Centrifuge the tube, and discard the collection tube with the flowthrough. To amplify the transposed DNA fragments, prepare the PCR master mixture without adding a unique Ad2. n barcoding primer.
Run the PCR. Run the quantitative PCR or qPCR. Check the amplification curves and identify the number of additional PCR cycles needed by estimating the number of cycles that reached approximately 35%of the maximum.
Use the calculations to run the PCR for the remaining 45 microliters of the PCR reaction. Using the earlier procedures, elute the amplified DNA fragments with 20 microliters of buffer EB.Transfer the eluted DNA to a new PCR tube and adjust the volume to 100 microliters by adding 80 microliters of buffer EB.Next, add 55 microliters of SPRI beads and mix by pipetting. After 5 minutes of incubation at room temperature, separate it on a mini magnetic stand for 5 minutes.
Once done, transfer 150 microliters of the supernatant to a new PCR tube, add 95 microliters of SPRI beads, and mix by pipetting. Incubate the reaction for 5 minutes before separating the beads on the mini magnetic stand. Discard the supernatant carefully and wash the beads for 1 minute using 200 microliters of 70%ethanol.
Repeat two more washes. After the final wash, centrifuge the tubes at 1000 G for 1 minute. Pipette out the remaining ethanol and dry the pellet at 37 degrees Celsius for 2 minutes on a PCR machine with the lid open.
Once dried, resuspend the pellet in 20 microliters of buffer EB by pipetting and incubate the tube for 5 minutes at room temperature. Separate the beads for 5 minutes on the mini magnetic stand before transferring 18 microliters of the supernatant containing the final library to a new 1.5 milliliter tube. mCherry-labeled and GFP-labeled adipocyte nuclei were distinguishable from the other cell types nuclei isolated from the adipose tissues of an Adipoq-NuTRAP mouse.
Depending on the type of adipose depot, the difference is of approximately 50%in eWAT, 30%in iWAT, and 65%in BAT were observed in the adipocyte fractions. The samples requiring more than 15 PCR cycles indicated low-quality samples. The size distribution analysis of the ATAC-seq libraries demonstrated multiple peaks corresponding to the nucleosome-free region, or NFR, and mono, di, and multi-nucleosomes with average sizes of approximately 500 to 800 base pairs.
Poor quality samples typically showed mostly NFR with no or few nucleosomal peaks. The quality check tests by the qRT-PCR analysis showed 10 to 20 fold enrichments with the positive genomic elements near adipocyte marker genes such as Adipoq, Fabp4, Plin1, and Pnpla2, while no enrichment was shown with the negative control. The enrichment with the thermogenic gene Ucp1 was observed specifically in BAT.
The mitochondrial reads were less than 2%and the fraction under the peaks was approximately 18%to 44%Multiple strong peaks with high signal-to-noise ratios were revealed near adipocyte marker genes such as Adipoq, Fabp4, and Plin1 from all the adipose depots. Strong ATAC-seq peaks were observed at the brown adipocyte marker Ucp1 locus in the BAT sample. The most critical factor for this protocol is nucleus quality.
It is important to use high-quality tissue samples and to gently handle the nuclei throughout the experiment, especially during resuspension after centrifugation and sorting. It's important to remember this protocol relies on transgenic reporter mice with nuclear labeling. NuTRAP mice were used here, but any similar system can be used.
With our FACS data, one can perform computational multiple analysis to find key transcriptomic factors that regulate gene expression in biological settings.
