Single-cell RNA sequencing is a powerful tool for studying transcriptional profiles of cells, particularly in heterogeneous tissue, such as the central nervous system. This protocol provides a method for isolating nuclei for single-nucleus RNA sequencing. By using nuclei instead of cells, this method can be applied to difficult-to-dissociate tissues, as well as frozen tissue.
Furthermore, this method can be applied to study cell type-specific changes in cells following disease or injury. Begin this procedure with mouse euthanasia, as described in the text protocol. Next, make an incision along the length of the body to expose the inner organs.
Eviscerate the mouse by pulling the inner organs from the body cavity using forceps. Do not use paper towels to clean the area or to remove organs, as this may introduce contaminants. Using scissors, cut the vertebral column between the L2 and L3 spinal vertebrae.
To eject the spinal cord, fit a three milliliter syringe containing ice-cold PBS with a 25 gauge 1/4 inch needle. Place the tip of the needle into the sacral end of the vertebral column. Use two fingers to pinch the vertebrae to create a tight seal around the tip of the needle, and press down on the plunger to eject the spinal cord rostrally.
Place the spinal cord in a Petri dish with ice-cold PBS. If only the lumbar portion of the cord is required, trim away the sacral and thoracic portions of the spinal cord. At this point, freeze the tissue and store at minus 80 degrees Celsius, or use immediately for either detergent mechanical cell lysis as described here, or hypotonic mechanical lysis as described in the text protocol.
To determine detergent mechanical cell lysis, place the lumbar spinal cord in a pre-chilled Dounce homogenizer and add 500 microliters of pre-chilled detergent lysis buffer. Dounce with five strokes of the loose pestle, A, and then five to 10 strokes of the tight pestle, B.Avoid lifting the homogenizer outside of the lysis solution in between strokes, and avoid introducing bubbles. Now, place a 40 micron strainer over a pre-chilled 50 milliliter conical tube and pre-wet with one milliliter of low sucrose buffer.
Add one milliliter of low sucrose buffer to the Dounce homogenizer containing the crude nuclei in the lysis buffer, and mix gently by pipetting two to three times. Pass the crude nuclei prep over the 40-micron strainer, into the pre-chilled 50 milliliter conical tube. Pass an additional one milliliter low sucrose buffer over the 40 micron strainer, bringing the final volume to three milliliters of the low sucrose buffer and 500 microliters of the lysis buffer.
Repeat these steps if combining multiple cords, cooling in the same conical tube. Then, centrifuge the sample at 3200 times G for 10 minutes at four degrees Celsius. Once the centrifugation is complete, decant the supernatant.
We suspend the palette using three milliliters of low sucrose buffer. Gently swirl to remove the pellet from the wall to facilitate the resuspension. After letting the sample sit on ice for two minutes, transfer the suspension to an Oak Ridge tube.
Using the homogenizer at setting one, homogenize the nuclei in low sucrose buffer for 15 to 30 seconds, keeping the sample on ice. Then, use a serological pipette to layer 12.5 milliliters of density sucrose buffer, underneath the low sucrose buffer homogenate, taking care not to create a bubble that disrupts the density layers. Centrifuge the tubes at 3200 times G, for 20 minutes at four degrees Celsius.
Once the centrifugation is complete, immediately decant the supernatant in a flicking motion. While attempting this procedure, it's important to remember to immediately remove the Oak Ridge tube from the centrifuge, and quickly decant the supernatant. Using 100 microliters to one milliliter of resuspension solution, we suspend the nuclei remaining on the wall.
Avoid the myelin frown that remains with the detergent-based preparation. Failure to do these steps quickly can result in a nuclei preparation with excess cellular debris, or myelin, which can impede downstream single-nucleus RNA sequencing. Filter the nuclei through a 30 to 35 micron pore size strainer and collect in a pre-chilled tube.
Determine the nuclei yield, using a hemocytometer to count nuclei under a 10 x objective, before proceeding to single-nucleus RNA sequencing, as described in the text protocol. Bright field and epifluorescent images of nuclei, isolated using the detergent mechanical lysis protocol, are shown at 10 x. These nuclei are isolated in an unbiased manner, allowing for the study of endogenous gene expression profiles at the level of a single cell.
Shown here, is a representative tSNE plot of single-nucleus RNA sequencing, from a mouse lumbar spinal cord, using a massively parallel droplet encapsulation platform. Nuclei isolated from fresh or frozen tissue can be used for sequencing on both commercial and academic platforms. This technique enables researchers to explore transcriptional profiles at the single-cell level, using difficult-to-dissociate tissues, such as the spinal cord, or even frozen tissue, such as biobank material.
While attempting this procedure, it's important to reduce the amount of time between euthanasia and cellular lysis. Furthermore, it is important to minimize bubbles introduced into solutions from cellular lysis to resuspension, and to buy pet nuclei with care. Following this procedure, nuclei can be used for alternative applications, such as facs sorting, in order to isolate specific cell types.
