Cell-Specific Paired Interrogation of the Mouse Ovarian Epigenome and Transcriptome

This method provides a technique to isolate nucleic acids from specific ovarian cell populations from the same mouse without the need for cell sorting. This technique can help identify how specific cell populations are changing under a variety of conditions. For example, with aging.

The main advantage of this technique is that it allows for paired epigenomic and transcriptomic analysis from a specific ovarian cell type without the need for cell sorting. This significantly increases the throughput, decreases the cost, and eliminates the need for specialized equipment when performing cell type-specific analysis from ovarian tissue. Here, our goal is to identify the cell type-specific changes that occur with ovarian aging prior to reproductive senescence, which has implications in treating female infertility.

This method can be applied to any cell type across body systems for which a cell type-specific Cre driver is available. The most difficult part of this technique is choosing an appropriate Cre line for your cell type of interest. Extensive validation of the cell specificity is recommended.

To begin, collect one ovary from the euthanized mouse in 500 microliters of nuclei lysis buffer and chop the ovary into eight parts within the buffer using self-opening micro scissors. Transfer the minced ovary and the lysis buffer into a glass dounce homogenizer on ice and homogenize the ovary 10 to 20 times with the loose pestle A.Add 400 microliters of lysis buffer to the dounce homogenizer washing pestle A.And then homogenize the ovary with tight pestle B 10 to 20 times. Transfer the homogenate to a two-milliliter round bottom tube and centrifuge at 200 g for 1.5 minutes at four degrees Celsius to remove the undissociated tissue and blood vessels.

After pre-wetting a 30-micrometer cell strainer with 100 microliters of lysis buffer, filter the supernatant through the strainer into a 15-milliliter conical tube. Transfer the nuclei containing mixture to a two-milliliter round bottom tube. Centrifuge the sample at 500 g for five minutes at four degrees Celsius and discard the supernatant.

Resuspend the nuclei pellet in 250 microliters of ice cold lysis buffer by vortexing briefly at moderate to high speed. Bring the volume up to 1.75 milliliters by adding 1.5 milliliters of lysis buffer. Mix gently and rest the nuclei suspension on ice for five minutes.

Pellet the nuclei by centrifugation at 500 g for five minutes at four degrees Celsius. And carefully discard the supernatant without disturbing the nuclei pellet. Resuspend the pellet in 200 microliters of ice cold storage buffer and vortex briefly to completely resuspend the nuclei pellet.

Reserve 10%of the resuspended pellet as an input nuclei sample for downstream analysis. Take the resuspended nuclei pallet and make up the volume to two milliliters with ice cold nuclear purification buffer, or NPB. Add 30 microliters of resuspended beads for each sample into a two-milliliter round bottom tube.

Then add one milliliter of NPB and resuspend well by pipetting. Place the tube on the magnetic rack for one minute to separate the beads. Discard the supernatant and remove the tube from the magnet.

Resuspend the washed magnetic beads in one milliliter of NPB. Repeat the wash step for a total of three washes. And after the final wash, resuspend the beads in the initial volume of NPB.

Add 30 microliters of the washed beads to two milliliters of the nuclear suspension and resuspend the beads nuclei mixture well by pipetting and gently inverting the tube. Place the tubes on a rotating mixer in a cold room or refrigerator for 30 minutes at low speed and incubate the input samples without beads in the same conditions. Place the tubes with the nuclei suspension and beads on the magnet for three minutes to separate the biotinylated nuclei bound to streptavidin beads from the negative fraction of nuclei.

Remove the supernatant. Pass the negative fraction to a fresh two-milliliter tube and reserve it on ice. For each positive fraction tube, remove the tube from the magnet and resuspend the contents in one milliliter of NPB.

Place the tube on the magnet for one minute and discard the supernatant. Resuspend the positive fraction bead nuclei mixture in 30 microliters of NPB. Transfer the ovary and 100 microliters of homogenization buffer into a glass dounce homogenizer and homogenize 10 to 20 times with the loose pestle A.Add 400 microliters of TRAP homogenization buffer to washing pestle A and transfer the homogenate to a two-milliliter round bottom tube.

Wash the homogenizer with an additional one milliliter of TRAP homogenization buffer and transfer the washed volume to the two-milliliter round bottom tube. Centrifuge the homogenate at 12, 000 g for 10 minutes at four degrees Celsius. And transfer the cleared supernatant to a fresh two-milliliter round bottom tube.

After discarding the pellet, transfer 100 microliters of the cleared homogenate to a fresh two-milliliter round bottom tube and reserve it as input on ice. Incubate the remainder of the cleared homogenate with five micrograms per milliliter anti-GFP antibody for one hour at four degrees Celsius while rotating in an end-over-end mixer. Incubate the input sample in the same condition.

Transfer 50 microliters of the protein G magnetic beads for each sample into a two-milliliter round bottom tube. Add 500 microliters of low-salt wash buffer to the beads and pipette gently to mix. Place the tube into a magnetic stand for one minute to collect the beads against the side of the tube.

After discarding the supernatant, remove the tube from the magnetic stand and add one milliliter of low-salt wash buffer to the tube. Pipette gently to mix. Again, place the tube into the magnetic stand and repeat the step for a total of three washes.

After the final wash, resuspend the beads in the initial volume of low-salt wash buffer. Transfer the resuspended washed beads to the antigen sample antibody mixture and incubate at four degrees Celsius overnight while rotating in an end-over-end mixer. Incubate the input samples overnight in the equivalent conditions.

After that, separate the positive fraction or the magnetic beads with the target ribosomes RNA from the negative fraction or the supernatant using the magnetic stand for 2.5 to 3 minutes. Aspirate the negative fraction. Place it in a fresh two-milliliter round bottom tube, and set it aside on ice.

Add 500 microliters of high-salt wash buffer to the tube with the positive fraction and gently pipette to mix. Place the tube on a magnetic stand maintained on ice for one minute to separate the beads. After discarding the supernatant, remove the tube from the magnet and repeat the step for a total of three washes.

After the final wash, resuspend the beads in 350 microliters of RNA lysis buffer supplemented with 3.5 microliters of 2-mercaptoethanol. Incubate the tubes at room temperature while mixing for 10 minutes at 900 RPM in a digital shaker. Separate the beads from the solution that now contains the target ribosomes RNA with a magnetic stand for one minute at room temperature.

Collect the eluted positive fraction in a fresh 1.5 milliliter tube. Immunofluorescence imaging was performed on the paraffinized ovaries of Cyp17-Cre positive NuTRAP flox and Cyp17-Cre negative NuTRAP flox mice. EGFP protein was stained using goat anti-GFP primary antibody and Alexa 488 donkey anti-goat secondary antibody, and the nuclei was stained with DAPI.

Images were taken on a fluorescence microscope at 20x magnification. The input and TRAP positive fraction RNA was isolated from Cyp17-Cre positive NuTRAP flox mouse ovaries and sequenced in a paired end manner. The principal component analysis of all the expressed genes showed a separation of the TRAP input and positive fractions in the first component.

The volcano plot of differentially expressed genes between the input and positive fractions is shown here. The comparison of the TRAP positive fraction to the input showed an overall enrichment of the stroma/theca cell marker genes and the depletion of the oocyte, granulosa, immune, and smooth muscle cell marker genes in the TRAP positive fraction. After the first centrifugation of the nuclear suspension to remove undissociated blood vessels, the nuclei are in the supernatant.

Make sure to keep the supernatant and discard the pellet at this point. Following the intact method, several epigenomic techniques can be conducted, including assays for chromatin accessibility or DNA modifications among others. Following the TRAP method, RT-qCPR or RNA sequencing can be conducted to assess the translatome.

This is the first application of the NuTRAP method in the mouse ovary. This allows for high throughput epigenomic and transcriptomic analysis from specific ovarian cell types, which may be used to study ovarian aging or cancer.