Method of Microglial DNA-RNA Purification from a Single Brain of an Adult Mouse

Microglia, the resident brain immune effectors cells, show dynamic activation level changes for most neuropsychiatric diseases, reflecting their complex regulatory function and potential as a therapeutic target. Emerging single-cell molecular biology studies are used to investigate the genetic modification of individual cells to better understand complex gene regulatory pathways. Although multiple protocols for microglia isolation from adult mice are available, it is always challenging to get sufficient purified microglia from a single brain for simultaneous DNA and RNA extraction for subsequent downstream analysis. Moreover, for data comparison between treated and untreated groups, standardized cell isolation techniques are essential to decrease variability. Here, we present a combined method of microglia isolation from a single adult mouse brain, using a magnetic bead-based column separation technique, and a column-based extraction of purified DNA-RNA from the isolated microglia for downstream application. Our current method provides step-by-step instructions accompanied by visual explanations of important steps for isolating DNA-RNA simultaneously from a highly purified microglia population.


Introduction
Microglia, the brain's resident immune cells, account for 5-12% of total cell population in the mouse brain and play a crucial role in adult brain neurogenesis, neuroinflammation, and overall brain homeostasis [1]. This multifunctional brain cell vastly depends on environmental stimuli to maintain its homeostatic phenotype [2] and immediately adjusts its functional profile based on the physiological or pathophysiological needs of the individual. Microglia mediate crosstalk between the brain and immune system, participating in the pathogenesis of neurodegenerative disorders and protecting against invading pathogens [3,4]. Characterization of the inflammatory profiles associated with crucial epigenetically regulated genes and connected signaling pathways in microglia are therefore significant in identifying potential therapeutic targets contributing to the pre-clinical progression of neurodegenerative disorders. Crosstalk between microglia and immune cells like major histocompatibility complex class II (MHC class II) and CD74 have recently been studied in multiple sclerosis (MS) pathology [5], which has increased our understanding of chronic inflammation observed in MS. Microglial genetic defects in neurodegenerative disorders may have a comprehensive impact on disease-specific pathology. Modulation of microglial function by gene editing on the whole brain or specific brain regions could hold promise for therapeutic targets in many neurodegenerative and neuropsychiatric diseases.
Accumulating evidence suggests that differences in DNA isolation methods could have a significant impact on downstream applications, especially relative mitochondrial DNA (mtDNA) abundance in whole-genome sequencing [6,7]. Similarly, differences in RNA isolation methods severely affect relative transcript abundance in RNA sequencing analysis [8,9].  Table 2 shows the detailed information of the equipments.   Table 3 shows the detailed information of the freshly prepared solutions.

2.
Perfuse the mice transcardially (through left ventricle) with 10-15 mL of ice-cold 1 × DPBS with calcium and magnesium before harvesting the brain.
(Note: Transcardiac perfusion eliminates circulatory CD11b positive cells from the brain; Therefore, continue perfusion till the last heartbeat for complete elimination.)

3.
Cut the skull upward along the sagittal suture starting from the brain stem, peel away two halves of the skull and scoop out the whole brain.
(Note: If transportation is needed, transfer the whole brain in a 15/50 mL sterile falcon tube containing ice-cold 1 × DPBS with calcium and magnesium placed in an ice box.)

4.
Place the brain segment into a sterile Petri dish on ice and wash 2-3 times with 1 mL of ice-cold 1 × HBSS without calcium and magnesium. 5.
Apply a sterile, sharp knife and forceps to mince the whole brain into multiple (10-15) slices on the same Petri dish, containing 1 mL of ice-cold 1 × HBSS without calcium and magnesium. 6.
Transfer all minced brain slices (for a single brain) into a 15 mL Falcon tube containing 1950 µL MACS Enzyme Mix-1/EM-1 (1900 µL Buffer Z + 50 µL Enzyme P) prewarmed at 37 • C for 5 min to aid in tissue dissociation step in Figure 1. 7.
Incubate the sample mixture in a water bath at 37 • C for 15 min with continuous shaking at a speed of 50 revolutions per minute (r.p.m.). 8.
At the end of incubation, agitate the aggregated tissue slices using a flame polished glass Pasteur pipette to make a single cell suspension (pipet up and down approximately 20 times or until all tissues move freely up into the Pasteur pipette).

Part 2: Debris Removal (40 min)
20. Resuspend cell pellets gently by pipetting with the appropriate volume (3100 μL/single brain) of ice-cold 1 × DPBS, without calcium and magnesium, and transfer cell suspension into a new sterile 15 mL conical tube. Do not vortex. 21. Add the appropriate volume (900 μL/single brain) of cold MACS debris removal solution into the resuspended sample and mix gently by pipetting 10-12 times as outlined in Figure 2. 22. Overlay 4 mL of ice-cold 1 × DPBS without calcium and magnesium very gently above the cell suspension to make a transparent gradient.
(Note: Start overlaying by holding the tube at a 45° angle and slowly bring the tube back to a vertical position as more DPBS is added on top of the layer of debris removal solution. Pipet very slowly to ensure that the two phases are not mixed.) 23. Centrifuge at 1000× g for 10 min at 4 °C with maximum acceleration and full brake.
(Note: After centrifugation, remove the tube from the rotor carefully so as not to agitate the three different phases (top liquid, middle solid debris, and bottom liquid). Supplemental Figure S1 shows the pre-and post-centrifugation gradients.) 24. Aspirate the two top phases (top liquid and solid interphase) completely and discard them.
(Note: Work quickly, as the solid debris interphase gradually settles down over time. Gently insert the pipette near the side of the solid interphase, then let the pipette tip touch

9.
Add 30 µL of MACS Enzyme mix-2/EM-2 (20 µL Buffer Y + 10 µL Enzyme A) into the agitated sample mix. Mix by pipetting; do not vortex or invert the tube. 10. Incubate the sample mix in a water bath at 37 • C for 10 min with continuous shaking at a speed of 50 r.p.m. 11. At the end of incubation, agitate the sample mixture 20-25 times using a 1 mL pipette. 12. Add DNase I (5 U/mL) into the agitated sample mix. Mix by pipetting; do not vortex or invert the tube. 13. Incubate the sample mix in a water bath at 37 • C for 10 min with continuous shaking at a speed of 50 r.p.m. 14. At the end of incubation, to completely stop the digestion reaction, dilute the tissue sample with 2 mL of ice-cold 1 × HBSS without calcium and magnesium and incubate on ice for 5 min. 15. Centrifuge tissue samples quickly at 800-1000× g for 10-15 min at room temperature (20-25 • C), decant the supernatant, and collect the cell pellet. 16. Resuspend cell pellets in 2 mL of ice-cold 1 × HBSS without calcium and magnesium. 17. Apply resuspended cell suspension directly into the middle of the pre-moisten MACS SmartStrainer (70 µm) placed on a sterile 50 mL Falcon tube.
(Note: Pre-wet/moisten the MACS SmartStrainer (70 µm) with 1-2 mL of ice-cold 1 × HBSS with calcium and magnesium and discard the flow-through. Improper or incomplete pre-wetting of the Strainer filter can cause cell stacking and sticking around the upper side of the Falcon tube surrounding the filter and increase cell loss.) 18. Apply 7 mL of ice-cold 1 × HBSS with calcium and magnesium on the MACS Smart-Strainer (70 µm). Rinse the old Falcon tube carefully by pipetting up and down with 1 mL 1 × HBSS with calcium and magnesium, and add it directly to the MACS SmartStrainer to prevent any cell loss. 19. Discard the MACS SmartStrainer (70 µm) and centrifuge cell suspension at 600-700× g for 10 min at 4-8 • C. Carefully remove the supernatant by vacuum aspiration or by pipetting.

Part 2: Debris Removal (40 min)
20. Resuspend cell pellets gently by pipetting with the appropriate volume (3100 µL/single brain) of ice-cold 1 × DPBS, without calcium and magnesium, and transfer cell suspension into a new sterile 15 mL conical tube. Do not vortex. 21. Add the appropriate volume (900 µL/single brain) of cold MACS debris removal solution into the resuspended sample and mix gently by pipetting 10-12 times as outlined in Figure 2. 22. Overlay 4 mL of ice-cold 1 × DPBS without calcium and magnesium very gently above the cell suspension to make a transparent gradient.
(Note: Start overlaying by holding the tube at a 45 • angle and slowly bring the tube back to a vertical position as more DPBS is added on top of the layer of debris removal solution. Pipet very slowly to ensure that the two phases are not mixed.) 23. Centrifuge at 1000× g for 10 min at 4 • C with maximum acceleration and full brake.
(Note: After centrifugation, remove the tube from the rotor carefully so as not to agitate the three different phases (top liquid, middle solid debris, and bottom liquid). Supplemental Figure S1 shows the pre-and post-centrifugation gradients.) 24. Aspirate the two top phases (top liquid and solid interphase) completely and discard them.
(Note: Work quickly, as the solid debris interphase gradually settles down over time. Gently insert the pipette near the side of the solid interphase, then let the pipette tip touch the solid interphase and aspirate the solid waste gently, followed by the top liquid phase removal.) 25. Fill the tube with the appropriate volume (~11 mL) of ice-cold 1 × DPBS without calcium and magnesium to a final volume of 14 mL. 26. Gently invert the tube three (3×) times. Do not vortex! 27. Centrifuge the tube at 1000× g for 10 min at 4 • C with maximum acceleration and full brake.
Methods Protoc. 2021, 4, x FOR PEER REVIEW the solid interphase and aspirate the solid waste gently, followed by the top liquid removal.) 25. Fill the tube with the appropriate volume (~11 mL) of ice-cold 1 × DPBS witho cium and magnesium to a final volume of 14 mL. 26. Gently invert the tube three (3×) times. Do not vortex! 27. Centrifuge the tube at 1000× g for 10 min at 4 °C with maximum acceleration an brake.
# During centrifugation, make a fresh PB buffer (e.g., 1 × DPBS without calciu magnesium with 0.5 × BSA and add 50 μL BSA into 9950 μL of 1 × DPBS to make of PB buffer). Before use, sterilize the filter first with PB buffer and then degas by uming at room temperature, as gas may clog the separation column and affect the q of separation. Keep the buffer cold at 2-8 °C. This is a preparatory procedure for S in Section 3.5., Part 5, to streamline the protocol and minimize wasted time.
# Prepare 1 × Red Blood Cell (RBC) removal solution for a single mouse br mixing 900 μL of sterile ddH2O with 100 μL of MACS 10 × RBC removal stock so This solution is for Step 28 in Section 3.3., Part 3.

Part 3: Red Blood Cell (RBC) Removal (30 min)
28. Aspirate supernatant completely and resuspend cell pellets carefully in 1 mL o 1 × RBC removal solution by pipetting up and down 10-15 times with a 1 mL p Do not vortex. 29. Incubate the cell mixture for 10 min in the refrigerator at 2−8 °C. 30. Add 10 mL of ice-cold PB buffer to the cell mixture to wash out the RBC re solution. 31. Centrifuge at 400× g for 10 min at 4 °C. Aspirate supernatant completely to elim RBC as shown in Figure 3. # During centrifugation, make a fresh PB buffer (e.g., 1 × DPBS without calcium and magnesium with 0.5 × BSA and add 50 µL BSA into 9950 µL of 1 × DPBS to make 10 mL of PB buffer). Before use, sterilize the filter first with PB buffer and then degas by vacuuming at room temperature, as gas may clog the separation column and affect the quality of separation. Keep the buffer cold at 2-8 • C. This is a preparatory procedure for Step 40 in Section 3.5., Part 5, to streamline the protocol and minimize wasted time.

Part 3: Red Blood Cell (RBC) Removal (30 min)
28. Aspirate supernatant completely and resuspend cell pellets carefully in 1 mL of cold 1 × RBC removal solution by pipetting up and down 10-15 times with a 1 mL pipette. Do not vortex. 29. Incubate the cell mixture for 10 min in the refrigerator at 2−8 °C. 30. Add 10 mL of ice-cold PB buffer to the cell mixture to wash out the RBC removal solution. 31. Centrifuge at 400× g for 10 min at 4 °C. Aspirate supernatant completely to eliminate RBC as shown in Figure 3.  (Note: Protect the magnetic beads and cell beads mixture from light. For optimal labeling, it is important to adjust the volume of PB buffer/beads ratio, for instance, with ≤1 × 10 7 total cells in 90 µL buffer, add 10 µL microglia micro-beads, and with ≥1 × 10 8 total cells in 180 µL buffer, add 20 µL microglia micro-beads.) 35. Place the tube (cell-beads mixture) in an End Over End shaker, keep the mix protected from light, and incubate for 15 min in the refrigerator at 2−8 • C.
(Note: At this stage, turn on the microcentrifuge machine and cool down the temperature to 4 • C for the next centrifugation steps.) 36. Add 1-2 mL of cold PB buffer into the cell-beads mixture per 10 8 cells and centrifuge at 300-350× g for 10 min at 4 • C to wash out the unlabeled beads. Aspirate supernatant completely. 37. Resuspend magnetically labeled cells (up to 5 × 10 7 cells/single mice brain) carefully in 500 µL of cold PB buffer and remove any bubbles if formed during resuspension. 36. Add 1-2 mL of cold PB buffer into the cell-beads mixture per 10 8 cells and centrifuge at 300-350× g for 10 min at 4 °C to wash out the unlabeled beads. Aspirate supernatant completely. 37. Resuspend magnetically labeled cells (up to 5 × 10 7 cells/single mice brain) carefully in 500 μL of cold PB buffer and remove any bubbles if formed during resuspension.
(Note: For higher cell numbers, scale up the PB buffer volume proportionately.) 38. Transfer 40 μL of the cell suspension to another sterile 1.5 mL tube from the 500 μL cells for later flow cytometric analysis (this cell fraction is designated as the total cells fraction or original cell fraction for FACS compensation analysis).  (Note: Pre-wet/moisten the pre-separation filter with PB buffer before use. For optimal separation, it is important to obtain a single-cell suspension before magnetic separation. Microglia has its territory about 15-30 µm wide, and the miniMACS MS columns are not suitable for particle sizes greater than 30 µm.)

Part 6: FACS Analysis (100 min)
(Before beginning; A. Centrifuge newly received antibodies at high speed to remove "aggregated immunoglobulin", a major source of nonspecific binding. B. Prepare fresh buffers: FACS staining buffer: Add 2×FBS in 1×DPBS without calcium and magnesium. FACS buffer: Add 5 mM EDTA to FACS buffer to avoid cell aggregation. C. Always stain on ice to prevent internalization of antigen-antibody complexes. A. Centrifuge newly received antibodies at high speed to remove "aggregated immunoglobulin", a major source of nonspecific binding. B. Prepare fresh buffers: FACS staining buffer: Add 2 × FBS in 1 × DPBS without calcium and magnesium. FACS buffer: Add 5 mM EDTA to FACS buffer to avoid cell aggregation. C. Always stain on ice to prevent internalization of antigen-antibody complexes. D. Keep stained sample protected from light and heat to avoid tandem degradation. E. Always include a live/dead (viability dye) staining dye to discriminate live cells. F.
Titrate optimal antibody concentration to maximize the signals to noise ratio. High antibody concentration can increase non-specific binding whereas lower concentration can decrease signal intensity. G. Carefully select the antibody conjugate. Fluorophores with the highest staining index (such as PE, APC) are best used for cells that have the lowest antigen expression or lower subsets, whereas dimmer fluorophores (PerCP, Alexa Fluor 405) are better suited for more highly expressed antigens or higher subsets. H. The combined volume of the cell sample and antibody should not exceed 100 µL.
High concentration or dilution may affect staining efficiency. I.
For multiple antigens staining, make an antibody cocktail with FACS stain buffer and then add equally to the designated cell suspension.)

2.
Distribute 40 µL of the "total cells fraction" or "original cell fraction" from Step #38 to another four (4) FACS Falcon tubes (individual tubes labeled as Unstained, CD11b-APC single stain, live/dead-violet 510 single stain, and all stain) and adjust each individual tube's final volume to 100 µL with 90 µL of FACS stain buffer.
(Note: At this stage, take out the live/dead staining dye vial (Tonbo Bioscience, Ghost Dye, Violet #510) from the −20 • C freezer, keep it from light, and allow it to equilibrate to room temperature. Before use, quickly spin Ghost dye vial.)

3.
Wash the cells once in 1 mL of FACS stain buffer and centrifuge at 350-400× g for 10 min at 20-25 • C. Resuspend cells in 90 µL of FACS stain buffer.
(Note: Resuspend up to 10 7 cells per 100 µL of FACS stain buffer. For higher cell numbers, scale up the volume of all reagents accordingly, e.g., for 2 × 10 7 cells, and double the volume of suspension buffer, antibody concentration, and washing buffer.)
Incubate in 2-8 • C freezer for 30 min in the dark. 6.
Wash cells once in 1 mL of FACS stain buffer and centrifuge at 350-400× g for 10 min at 20-25 • C. Resuspend cells in 100 µL of FACS stain buffer. 7.
(Note: Fluorophore-conjugated antibodies may bind to cell surface Fc receptors and contribute to non-specific staining. It is therefore recommended to block Fc-receptors (anti-mouse CD16/CD32 monoclonal antibody) before intended surface receptor (CD11b) staining.)
Add 5 µL of anti-mouse and human CD11b antibody (Tonbo Bioscience) directly to the pre-incubated cell suspension in the presence of the CD16/CD32 antibody and vortex to mix.
(Note: The anti-mouse CD16/CD32 (Fc shield) antibody does not need to be washed away before staining cells with the CD11b antibody.) 10. Incubate cell suspension with the CD11b plus Fc shield antibody then mix in a 2-8 • C freezer for 30 min in the dark.
(Note: Longer incubation time, higher temperature, and incubation on ice may affect cell staining.) 11. Wash cells twice in 1 mL of FACS buffer and centrifuge at 350-400× g for 10 min at 20-25 • C.
(Note: Aspirate supernatant carefully, leaving around a 100 µL buffer with the cell pellet covering the bottom of the tube for the first wash, followed by complete aspiration of the supernatant in the second wash.) 12. Aspirate supernatant completely via vacuum aspiration and resuspend cell pellets (briefly vortex to dissociate the cell pellet) in a suitable amount (300-400 µL) of FACS buffer for analysis by flow cytometry (FACS) immediately, or store it in a 4 • C freezer for short-term storage. Supplemental Figure S2 shows the gating strategy and associated purity of isolated microglial populations.

Part 7: Proteomic Analysis (24 h)
1. Add 150 μL of RIPA extraction buffer, supplied with protease inhibitor, to microglia cell pellets from Step 49 after the 1 × DPBS wash and resuspend by pipetting up and down with a 200 μL pipette. 2. Homogenize cells on ice (to protect from overheating, proteases work best at high temperature) by sonication (total 3-6 pulse, 5-s intervals among individual pulses) to get a uniform, opaque solution with microscopic bubbles. 3. Immediately incubate the sample on ice for 10 min and then centrifuge at 15,000× g for 15 min at 4 °C. 4. Transfer supernatant to a fresh sterile microcentrifuge tube and keep a record of the transfer volume. 5. Quantify protein concentration by the Bradford assay or similar protein assay methods and add 3 × protein loading buffer, considering the total transfer volume. 6. Incubate the sample at 95 °C for 5 min to denature the protein, spin briefly, and use for protein loading or store at −80 °C for later use. 7. Load an equal amount of the sample (30 μg for GAPDH and IBA1 and 40 μg for GFAP and NeuN) into 10 × SDS-PAGE gel (1.5 mm thick) and run at 100 V. Load protein Homogenize cells on ice (to protect from overheating, proteases work best at high temperature) by sonication (total 3-6 pulse, 5-s intervals among individual pulses) to get a uniform, opaque solution with microscopic bubbles.

3.
Immediately incubate the sample on ice for 10 min and then centrifuge at 15,000× g for 15 min at 4 • C.

4.
Transfer supernatant to a fresh sterile microcentrifuge tube and keep a record of the transfer volume.

5.
Quantify protein concentration by the Bradford assay or similar protein assay methods and add 3 × protein loading buffer, considering the total transfer volume.

6.
Incubate the sample at 95 • C for 5 min to denature the protein, spin briefly, and use for protein loading or store at −80 • C for later use. 7.
Load an equal amount of the sample (30 µg for GAPDH and IBA1 and 40 µg for GFAP and NeuN) into 10 × SDS-PAGE gel (1.5 mm thick) and run at 100 V. Load protein ladder (page ruler pre-stained protein ladder) to identify the protein band according to molecular weight. 8.
Activate the PVDF membrane by pre-wetting in methanol for 10 min followed by 10 min of wetting in the transfer buffer. 9.
Disassemble gel tank, carefully remove the gel, and place on the PVDF membrane, then reassemble the gel-sponge-membrane sandwich in the wet transfer tank (Bio-Rad mini protean tetra cell) containing transfer buffer and run the power supply at 300 mA for 2 h to completely transfer the protein to the PVDF membrane. 10. Disassemble the protein transfer tank, carefully remove the PVDF membrane, and block the membrane with the vacuum filtered blocking buffer (3% w/v BSA in 1 × TBST) to avoid a high background during chemiluminescence detection due to non-specific binding of the primary and secondary antibody of interest. 11. Incubate the PVDF membrane with the primary antibody of interest (diluted in 5% w/v BSA in 1 × TBST) in a GenHunter Western blot container placed on a platform shaker overnight at 4 • C (cover the membrane completely with antibody solution). 12. Remove the primary antibody after incubation, wash the membrane 3-4 times with TBST (5 min for each time), and incubate with the secondary antibody (diluted in blocking buffer) for 1 h at room temperature. 13. Remove the secondary antibody after incubation and wash the membrane 3-4 times with TBST (5 min for each time). 14. Detect the protein band by applying an equal volume of chemiluminescence solution E and F in a chemiluminescence detector (Azure biosystem, c600). Loosen the cell pellet by flicking the tube 2-3 times.

4.
Transfer the lysate directly into a QIAshredder spin column placed on a 2 mL tube. Centrifuge at 20,000× g for 2-3 min.

5.
Transfer the supernatant carefully (leave any solid pellet or undissolved materials at the bottom) into a Qiagen AllPrep DNA spin column. Place in a 2 mL collection tube. Close the lid carefully so that it does not touch the liquid and centrifuge at ≥8000× g for 30 s. Use the flow-through for RNA extraction.

6.
Place the AllPrep DNA spin column in a new 2 mL blank collection tube and centrifuge at ≥8000× g for 30 s. After centrifugation, store the AllPrep DNA column at room temperature (15-25 • C) for immediate use or at 4 • C for later DNA purification.
Methods Protoc. 2021, 4, x FOR PEER REVIEW 14 of 18 9. Place the AllPrep DNA spin column in a new 2 mL blank collection tube and discard the old collection tube with the flow-through. Centrifuge at 20,000× g for 1 min. 10. Place the AllPrep DNA spin column into a new 1.5 mL DNases-free collection tube.
Add 15-25 μL of the appropriate volume of Buffer EB directly to the spin column membrane and close the lid carefully. Incubate at room temperature (15-25 °C) for 2-3 min followed by centrifugation at ≥8000× g for 1 min to elute the DNA. (Note: It is important to adjust the volume of 70% ethanol with the volume of flow through left after homogenization and DNA removal steps.) 12. Pipet up to 700 μL of the sample including any precipitate from Step 11 into the RNeasy spin column and place in a 2 mL collection tube. Close the lid carefully so that it does not touch the sample and centrifuge at ≥8000× g for 15 s. Remove the flowthrough by pipetting. Reuse the collection tube for the next step.
(Note: If the sample volume (flow-through plus 70% ethanol) exceeds 700 μL, centrifuge aliquots successively in the same RNeasy spin column. Remove the flow-through after each centrifugation.) 13. Add 700 μL buffer RW1 to the RNeasy spin column. Close the lid carefully so that it does not touch the sample and centrifuge at ≥8000× g for 15 s to wash the RNeasy spin column membrane. Remove the flow-through completely by pipetting. Reuse the collection tube for the next step.
[Note: Remove RNeasy spin column carefully from the collection tube after centrifugation so that the column does not contact the flow-through] 14. Add 500 μL buffer RPE to the RNeasy spin column. Close the lid carefully so that the column does not contact the flow-through and centrifuge at ≥8000× g for 15 s to wash the RNeasy spin column membrane. Remove the flow through completely. Reuse the collection tube for the next step. Add 500 µL buffer AW1 to the AllPrep DNA spin column from step 6. Close the lid carefully so that it does not touch the flow-through and centrifuge at ≥8000× g for 15 s. Discard the flow-through by pipetting. Reuse the DNA spin column for the next step. 8.
Add 500 µL of buffer AW2 to the AllPrep DNA spin column, close the lid carefully so that it does not touch the flow-through, and centrifuge at 20,000× g for 2 min to wash the DNA spin column membrane.
(Note: Remove RNeasy spin column carefully from the collection tube after centrifugation so that the column does not contact the flow-through.)

9.
Place the AllPrep DNA spin column in a new 2 mL blank collection tube and discard the old collection tube with the flow-through. Centrifuge at 20,000× g for 1 min. 10. Place the AllPrep DNA spin column into a new 1.5 mL DNases-free collection tube.
Add 15-25 µL of the appropriate volume of Buffer EB directly to the spin column membrane and close the lid carefully. Incubate at room temperature (15-25 • C) for 2-3 min followed by centrifugation at ≥8000× g for 1 min to elute the DNA. (Note: If the sample volume (flow-through plus 70% ethanol) exceeds 700 µL, centrifuge aliquots successively in the same RNeasy spin column. Remove the flow-through after each centrifugation.) 13. Add 700 µL buffer RW1 to the RNeasy spin column. Close the lid carefully so that it does not touch the sample and centrifuge at ≥8000× g for 15 s to wash the RNeasy spin column membrane. Remove the flow-through completely by pipetting. Reuse the collection tube for the next step.
[Note: Remove RNeasy spin column carefully from the collection tube after centrifugation so that the column does not contact the flow-through] 14. Add 500 µL buffer RPE to the RNeasy spin column. Close the lid carefully so that the column does not contact the flow-through and centrifuge at ≥8000× g for 15 s to wash the RNeasy spin column membrane. Remove the flow through completely. Reuse the collection tube for the next step. 15. Add 500 µL of buffer RPE to the RNeasy spin column. Close the lid carefully so that the column does not contact the flow-through and centrifuge at ≥8000× g for 2 min to further wash the spin column membrane. Remove the flow-through by pipetting.
Reuse the collection tube for the next step.
(Note: After centrifugation, remove the RNeasy spin column carefully from the collection tube so that the column does not contact the flow-through.) 16. Place the RNeasy spin column to a new 2 mL blank collection tube and discard the old collection tube with the flow-through. Centrifuge at 20,000× g for 1 min. 17. Place the RNeasy spin column in a new 1.5 mL RNase-free collection tube. Add 15-25 µL or an appropriate volume of RNase-free water directly to the spin column membrane. Close the lid gently and centrifuge at ≥8000× g for 1 min to elute the RNA as shown in Figure 8. Supplemental Figure S3 shows the quality control analysis of DNA-RNA isolated from purified microglia.  15. Add 500 μL of buffer RPE to the RNeasy spin column. Close the lid carefully so that the column does not contact the flow-through and centrifuge at ≥8000× g for 2 min to further wash the spin column membrane. Remove the flow-through by pipetting. Reuse the collection tube for the next step.
(Note: After centrifugation, remove the RNeasy spin column carefully from the collection tube so that the column does not contact the flow-through.) 16. Place the RNeasy spin column to a new 2 mL blank collection tube and discard the old collection tube with the flow-through. Centrifuge at 20,000× g for 1 min. 17. Place the RNeasy spin column in a new 1.5 mL RNase-free collection tube.  μL or an appropriate volume of RNase-free water directly to the spin column membrane. Close the lid gently and centrifuge at ≥8000× g for 1 min to elute the RNA as shown in Figure 8. Supplemental Figure S3 shows the quality control analysis of DNA-RNA isolated from purified microglia.

Results and Discussion
This combined method yielded purified microglia cells ranging between 3 × 10 5 and 5 × 10 5 cells from one adult mouse brain after double-column filtering, where the viability of purified microglia ranged between 70-80% of total cells and the purity ranged between 9 and 95%. Using Qiagen AllPrep DNA/RNA Mini kit (Step 8), total RNA yield ranged between 200 and 400 ng and genomic DNA yield ranged between 200 and 500 ng per one

Results and Discussion
This combined method yielded purified microglia cells ranging between 3 × 10 5 and 5 × 10 5 cells from one adult mouse brain after double-column filtering, where the viability of purified microglia ranged between 70-80% of total cells and the purity ranged between 9 and 95%. Using Qiagen AllPrep DNA/RNA Mini kit (Step 8), total RNA yield ranged between 200 and 400 ng and genomic DNA yield ranged between 200 and 500 ng per one adult mouse brain after double-column filtering. Single-column filtering resulted in a higher number of microglial cells, ranging between 3 × 10 5 and 6 × 10 5 , and higher cell viability, ranging between 80 and 90% of total cells, where the purity of isolated microglia was between 75 and 85%. The total RNA yield from single column filtering ranged between 250 and 600 ng and genomic DNA yield ranged between 300 ng and 1 µg. We have improved the number of purified cells from a single brain by calibrating the time and types of the brain tissue digestion system and adding an extra washing step (Step 49) for full functionality of the lysis buffer later in DNA-RNA isolation steps. Using the Qiagen AllPrep DNA/RNA/miRNA Universal kit following manufacturing instructions, DNA yield further increased to 800-1000 ng, where RNA yield increased to 500-800 ng for double-column filtering.
Purification of microglia following absolute enzymatic tissue dissociation protocol induces an abnormal gene expression signature in microglia that can mislead downstream analysis [22] and a combination of mechanical and enzymatic tissue dissociation methods was recommended very recently [23]. We used MACS ® Tissue Dissociation Kits with minor modifications that combine mechanical tissue dissociation with enzymatic treatment to obtain high yields of viable single cells as stated in Table 4 with preserved membrane integrity from hardly dissociated brain tissue. We used CD11b for microglia selection as it is expressed by both resting and activated microglia. Although, some recent studies [24,25] suggested TMEM119 as a more specific marker that can distinguish microglia from infiltrating macrophages in pathological condition. However, TMEM119 expression is absent in immature microglia [25] and changes in immature microglia are critical for downstream studies, as it can lead to persistent changes in microglial function, resulting in long-term neuronal dysfunction [26]. Moreover, sorting of microglia (FACS) with TMEM119 selection from a single mouse brain yields a smaller number of viable cells compared to MACS CD11b selection [27]. Table 4. Expected yields. This table summarizes the yield of microglia and subsequent DNA/RNA isolation using both single and double column filtering as well as the Qiagen mini and universal kits. The ranges represent the minimum and maximum results obtained from eight seven-month-old mice.

RNA (Universal)
This method yielded 500-800 µg of protein from one adult mouse brain and overcame the BSA contamination issue as evident by the immunoblotting images in Figure 9. BSA contamination in the extracted protein sample was minimized by a single 1 × DPBS wash (Step 49). Although we used a MACS enzymes system for tissue dissociation steps, we also tested a Collagenase-Dispase-DNaseI system that yields similar results. We have successfully used this method for microglia DNA-RNA isolation from a single, whole brain of an adult mouse. We believe it is possible to isolate microglia and microglial DNA-RNA from specific brain regions using this method, but optimization of the method, especially tissue dissociation and enzyme digestion, are highly recommended before use. wash (Step 49). Although we used a MACS enzymes system for tissue dissociatio we also tested a Collagenase-Dispase-DNaseI system that yields similar results. W successfully used this method for microglia DNA-RNA isolation from a single brain of an adult mouse. We believe it is possible to isolate microglia and microgli RNA from specific brain regions using this method, but optimization of the met pecially tissue dissociation and enzyme digestion, are highly recommended befo Figure 9. Purity of isolated mouse microglia population by proteomic analysis. Cell lysates CD11b positive and negative fraction were subjected to Immunoblotting. Protein band ca immunoblot analysis after probing with GAPDH (loading control), GFAP (astrocyte), IBA glia), and NeuN (Neurons) antibody. CD11b-positive cell lysates were positive for IBA1 tive for GFAP and NeuN, whereas CD11b-negative cell lysates were negative for IBA1 bu for GFAP and NeuN. Figure 9. Purity of isolated mouse microglia population by proteomic analysis. Cell lysates from the CD11b positive and negative fraction were subjected to Immunoblotting. Protein band captured in immunoblot analysis after probing with GAPDH (loading control), GFAP (astrocyte), IBA1 (microglia), and NeuN (Neurons) antibody. CD11b-positive cell lysates were positive for IBA1 but negative for GFAP and NeuN, whereas CD11b-negative cell lysates were negative for IBA1 but positive for GFAP and NeuN.

Conclusions
Our current method describes details of an efficient DNA-RNA isolation method from purified microglia from one adult mouse brain suitable for microglial transcriptomics and proteomics analysis. This method can also be used for any study requiring microglial single-cell transcriptomic profiling, DNA methylation analysis, proteomics analysis by Western blot, qRT-PCR, and immunophenotyping by flow cytometry in any number of adult mice.  Figure S3, Quality control analysis of isolated DNA-RNA from purified microglia cells. (a) Total RNA was isolated from purified microglia cells using the AllPrep DNA/RNA Mini Kit, RNA band intensities from wild type and transgenic mice (2) captured by RNA gel electrophoresis, corresponding RNA integration value, RIN calculated by peak area measurement. (b) Genomic DNA was isolated from purified microglia cells using the AllPrep DNA/RNA Mini Kit, genomic DNA band intensities from wild type and transgenic mice (2) captured by DNA gel electrophoresis, corresponding DNA integration value, DIN calculated by peak area measurement.