This method allows unbiased capturization of microglia gene expression at a single-cell level, which can help elucidate the molecular heterogeneity of microglia in a healthy and diseased brain. The main advantage of this technique is that it provides a detailed procedure for rapid isolation of microglia from different brain regions and shows how to efficiently generate play base the single-cell RA6 libraries for deep sequencing. To begin this procedure, prepare all needed buffers and solutions as outlined in the text protocol and chill them on ice.
After euthanizing and decapitating the mouse, use small scissors to cut open the skin on the head to expose the skull underneath. Then, cut through the sagittal suture, lambdoidal suture, and coronal suture. Use forceps to pull off both sides of parietal bone and interparietal bone without damaging the tissue and carefully move the brain into a dissection Petri dish containing medium A.Using a pre-chilled blade, cut the brain through the midline into two hemispheres.
Use number 55 forceps to separate the cerebellum from the cortical lobe and the brain stem. Then, use number 55 forceps to carefully dissect out the hippocampus and the striatum from the cortex and transfer each tissue into a separate collection Petri dish. First, use a razor blade to chop each brain region into fine pieces that are less than one cubic millimeter.
Using a one milliliter pipette with the tip cut off, transfer the tissue pieces into pre-chilled Dounce homogenizers. Homogenize the tissue by slowly twisting the piston in and out of the Dounce homogenizer for six to 10 full strokes until no visible chunks are present. Then, transfer the dissociated tissues into 50 milliliter tubes through 70 micrometer strainers.
Rinse each Dounce homogenizer and piston with the total of six milliliters of cold medium A then transfer the rinsing to a 15 milliliter tube for centrifugation. Centrifuge single-cell suspension at 400 times g and at four degrees Celsius for five minutes with a break at five. Rinse one large depletion column and three large selection columns in a magnetic separator with three milliliters of MCS.
When the centrifugation for the tissue samples is complete, pipette out and discard supernatant without disturbing the pellet. Resuspend the cells in MCS buffer that contains RNase inhibitor. Add 100 microliters of myelin removal beads to each tube containing re-suspended cells from the cortex and cerebellum and add 50 microliters of myelin removal beads to each of the tubes containing re-suspended cells from the hippocampus and striatum.
Incubate the tubes on ice for 10 minutes. After this, add MCS to the tube containing cortical cells to bring its volume up to two milliliters and to the remaining tubes to bring each of their volumes up to one milliliter. Once the columns are empty of rinsing buffer, load two milliliters of cortical cells onto the large depletion column and one milliliter of each other cell suspension onto separate large selection columns.
Next, wash the large depletion column ones with one milliliter of MCS buffer and wash each large selection column twice using one milliliter of MCS buffer for each wash. Filter the cells through 35 microliter strainer caps into round-bottom FACS tubes. Centrifuge these tubes at 400 times g and at four degrees Celsius for five minutes to pellet the cells.
Then, slowly pour out the supernatant and dab the edge of the tube on tissue paper. Resuspend the cells in each tube with 300 microliters of FACS buffer. First, add five microliters of mouse FC receptors block reagent to each tube.
Incubate on ice for five minutes. Then, add one microliter of CD45 PE size seven and one microliter of CD11B BV421 to each tube. Incubate the tubes on a shaker at room temperature for 10 minutes.
After this, add two milliliters of FACS buffer to wash. Centrifuge at 400 times g and at four degrees Celsius for five minutes to pellet the cells. Resuspend the cells in each tube with 400 microliters of FACS buffer containing one microliter of RNase inhibitor and 0.5 microliters of propidium iodide.
Following the standard FACS procedure, sort single live microglia with a 100 micrometer nozzle into 96-well PCR plates that contain four microliters of lysis buffer in each well. Briefly vortex the plates and spin them down using a bench top centrifuge. Store the plates at minus 80 degrees Celsius until library preparation.
When ready to proceed, thaw the plate on ice. Use a thermal cycler to perform a first transcription to generate cDNA and amplify the cDNA with an additional exonuclease digestion step as outlined in the text protocol. After this, purify the cDNA by adding 18 microliters of magnetic beads to each well.
Incubate the plate on a magnetic stand for five minutes. Then, remove the supernatant and wash the samples twice with freshly made 80%ethanol using 80 microliters of ethanol per well for each wash. To perform tagmentation, use a nanoliter pipetting machine to mix 0.4 microliters of each cDNA sample with 1.2 microliters of Tn5 tagmentation reagents from the library preparation kit in a 384-well plate at 55 degrees Celsius for 10 minutes.
To stop the tagmentation reaction, add 0.4 microliters of neutralization buffer at room temperature for five minutes. Next, at 384 indexes and 1.2 microliters of PCR mixed to the samples and amplify the libraries as outlined in the text protocol. Pool all of the individual libraries from the same 384-well plate together and use magnetic beads to purify the final pooled libraries.
In this study, microglia are isolated from the cortex, cerebellum, hippocampus, and striatum of an adult mouse's brain hemisphere. The prepared cell suspension is examined under a microscope by using trypan blue and a hemocytometer to estimate the yield, cell viability, and efficacy of myelin removal before performing antibody staining. Total cell counts at this point should be over 30, 000 for the cortex and over 5, 000 for the other tissues.
Over 90%of the cells should be viable with little myelin debris. A FACS machine is used to sort the microglia, which are typically CD45 low and CD11B positive. Successful isolation should generate over 80%microglia out of all live single-cells, at least for the cortical tissue.
Once individual microglia are captured into the lysis buffer, RNA is released and subsequently reverse transcribed to cDNA, which is then amplified for 23 cycles. As a capillary electrophoresis platform, the fragment analyzer and high-sensitive NGS fragment kits provide quick and accurate information about size distribution, as well as quantity of cDNA molecules present in each well of a 96-well plate. Samples showing a smear between 500 and 5, 000 base pairs and are above a certain concentration threshold can be used to make libraries.
The samples are sequenced to a depth of over one million raw reads per cell, which saturates the detection power of the single-cell RNA sequencing methodology. With a mapping rate of approximately 60%over 2, 000 genes can be detected per microglial cell. Previously published data generated from this isolation method is reproduced from independent experiments, demonstrating the sensitivity for detecting microglia specific genes across the sequenced population.
With the information of single microglia transcriptions, researchers can discover the unique cell populations in the context of their interest, and further study the functional relevance of these newly identified populations.
