This protocol describes the manual sorting procedure to isolate single fluorescently labeled neurons followed by in vitro transcription-based mRNA amplification for high-depth single-cell RNA sequencing.
This method can help solve a key obstacle in the field of single-neuron isolation and sequencing. Such as collecting specific population or a subpopulation of fluorescently labeled neuron from user-defined brain area. The main advantage of this technique is that it ensures the single neuron of interest is captured without contaminating debris.
It is also relatively gentle, which is important for fragile neuron types that die quickly when subjected to the fluid pressures of conventional FACS sorting. Start by preparing pulled glass microcapillaries with 10 to 15 micron exit diameter using a capillary puller. Use fine scissors to cut the tip of the capillary to get the desired board diameter.
Then use tubing connectors to attach a 120 to 150-centimeter length of flexible silicone tubing with 0.8 millimeter inside diameter to a 0.2 micron PVDF membrane syringe filter and a two-way tubing valve. Prepare 500 milliliters of chilled artificial cerebral spinal fluid or ACSF and oxygenate by bubbling 5%carbon dioxide balanced oxygen through an Airstone for 15 minutes or until the solution clears completely. Prepare three 150-milliliter beakers each containing 100 milliliters of ACSF.
To one, add a cocktail of activity blockers. To another, add Streptococcus fraction for protease to a final concentration of one milligram per milliliter. To the final beaker, add FBS to a final concentration of 1%and oxygenate with 5%carbon dioxide-balanced oxygen through an Airstone.
Finally, make three long-stemmed Pasteur pipettes with decreasing exit diameters by rolling them over an open flame. Dissect the brain from a euthanized mouse by opening the cranium with a small scissor and extracting the fresh brain using fine forceps without damaging the cortex. Place the brain in chilled and oxygenated ACSF leaving an Airstone attached to 5%carbon dioxide-balanced oxygen during the entire duration of the sectioning.
Position the brain on the vibratome chuck and cut coronal or sagittal sections at 300 micron thickness. Collect as many sections as needed to obtain a minimum of 10 to 50 labeled cells. Put slices on a cotton-meshed slice holder placed inside a beaker so they are bathed in oxygenated ACSF.
Move the slices into the beaker containing ACSF with activity blockers and block for 15 to 20 minutes at room temperature while bubbling oxygen using an Airstone. Move the slices to the beaker containing ACSF with the protease solution to perform mild digestion at room temperature. Following the digestion, wash out the protease by moving slices back to the beaker containing ACSF with activity blocker solution for five to 10 minutes at room temperature.
Keep bubbling oxygen. Next, prepare a 100-millimeter Petri dish containing ACSF with 1%FBS at room temperature and move individual sections into the disk for microdissection. Under a fluorescent dissections scope, use a pair of fine forceps to micro dissect areas and layers of interest having a minimum of 10 to 50 cells.
Using a Pasteur pipette, move the micro-dissected pieces to a 2-milliliter microcentrifuge tube containing approximately 0.8 milliliters of 1%FBS in ACSF solution. Titrate the dissected tissue in the micro-fuge tube at room temperature. Perform approximately 10 strokes with each of the flamed Pasteur pipettes starting with the biggest and ending with the smallest exit diameter.
Dispense the dissociated cells into a 100-millimeter Petri dish containing oxygenated ACSF and wait five to seven minutes for the cells to gradually settle. Observe the GFP and RFP signal under a dissection microscope. Identify debris by examining the morphology under bright-field illumination.
Once an area of cells with little debris has been identified, use the capillary and attach to tubing to pick cells by blocking the end of the tubing valve with the tongue. Then position the capillary close to the cells. Release the block on the capillary and quickly block again to capture the cell using capillary action.
Move the capillary to a 100-millimeter Petri dish with fresh oxygenated ACSF and gently blow into the tube while observing the capillary tip under fluorescence optics to dispense the cells into the dish. Repeat the collection procedure until 100 to 150 cells are collected making sure that contaminants, such as debris, are minimal in bright-field differential interference contrast optics. Finally, using a fresh micro-capillary pipette each time, choose a single cell and expel it in no more than 0.5 microliters into a 0.2 microliter micro-fuge tube containing one microliter of sample-collection buffer with primers.
Break the pipette tip in the micro-fuge tube to ensure that the cell stays in the collection buffer. Freeze the cells by putting the tubes on dry ice. Store in a minus 80-degree Celsius freezer until processing for RNA amplification.
GABAergic neurons were isolated from the cingulet cortex of the mouse brain. RNA amplified from the manually sorted neurons ranged between 200 to 4, 000 base pairs in size with a peak distribution slightly above 500 base pairs. After bead purification, the cDNA library is further size restricted by a second round of purification using beads to eliminate fragments less than 200 base pairs and for the peak at approximately 350 base pairs.
Sequencing showed a 4.8 times 10 to the 5th median or 6.9 times 10 to the 5th averaged mapped reads per cell. After duplicate RNA removal using UMIs, each single cell had a 1.0 times 10 to the 5th median or 1.4 times 10 to the 5th average unique reads per cell. In each single cell external RNA controls consortium, spike in RNA was used as an internal control to calculate absolute number of molecules added to the sample.
There was a linear relationship of input to observed counts with a slope of 0.92 and adjusted R square of 0.94. An average of around 10, 000 genes per single neuron were detected using this procedure with more than 95%of the single cells detecting more than 6, 000 genes. After its development, this technique paved the way for molecular biologists to explore the transcriptome of distinct well-characterized mouse cortical interneurons, and identified select gene categories that are responsible for encoding the cellular identity.
