Fluorescence-Activated Nuclei Negative Sorting of Neurons Combined with Single Nuclei RNA Sequencing to Study the Hippocampal Neurogenic Niche

Animal care and experimental procedures were performed in accordance with the guidelines of the Francis Crick Institute, as well as the UK Home Office guidelines and laws.

Figure 1: Preparation of a single nuclei suspension from the dissected DG of adult mice for snRNA-seq of non-neuronal populations. Flow diagram describing the main steps of the protocol that include dissection of mouse DG, preparation of a suspension of single nuclei, NeuN immunostaining, and negative NeuN-FANS-sorting before proceeding with snRNA-seq. Please click here to view a larger version of this figure.

1. Dissection of the DG (Timing: 15 min)

1. Prepare nuclei isolation media 1 and 2 (NIM1 and NIM2), homogenization buffer (HB), and Wash media (WM) (Supplemental Table 1). Place all the buffers, media, reagents, and tools on ice until needed. Put the Dounce homogenizer (see Table of Materials) on ice during preparation (minimum 1 h before the homogenization step).
   NOTE: NIM1 can be prepared and stored at 4 °C for up to 6 months. NIM2, HB, and WM should be freshly prepared.
   CAUTION: Manipulate DTT, the protease inhibitor and Triton X-100 with care. These compounds are skin and eye irritating, acutely toxic, and hazardous for the aquatic environment. While using these chemicals wear protective gloves, clothing, eye and face protection, wash hands thoroughly after handling, and avoid release to the environment.
2. Euthanize a 22-month-old male C57Bl/6J mouse by cervical dislocation following the Home Office Schedule 1 procedure²⁰.
   NOTE: See discussion for rationale regarding the use of 22-month-old mouse in this study. However, this protocol can be performed at any age across the lifespan.
3. Dissect the brain out from an euthanized mouse and transfer it into a 10 cm Petri dish filled with ice-cold 1x PBS (Figure 1). Place the Petri dish on ice. Remove the cerebellum using a scalpel and cut the brain in half between both the hemispheres (along the sagittal axis).
4. Fill in a new 10 cm Petri dish with ice-cold PBS and place it on ice. Transfer one half of the brain to the new Petri dish. Using binoculars, dissect out the DG and repeat this step to obtain the second DG from the second half of the brain.
   ​NOTE: These steps (steps 1.2-1.4) were adapted from a previously described procedure²¹. It is important to proceed as rapidly as possible at this stage to preserve the integrity of the cells.
5. Transfer the two DGs into the precooled Dounce homogenizer and add 1 mL of cold HB.

2. Tissue dissociation, single nuclei isolation, and anti-NeuN immunostaining (Timing: 2 h)

1. Homogenize the tissue with 10 strokes of the loose "A" pestle, followed by 15 strokes of the tight "B" pestle.
   NOTE: Dounce homogenization should be performed with the mortar on ice with gentle strokes to reduce heat caused by friction and foaming. All the buffers and equipment should be precooled and kept on ice during the procedure.
2. Transfer the homogenate into a prechilled 15 mL tube; rinse the Dounce homogenizer with 1 mL of cold HB and combine to the same tube. Add 3 mL of HB to the 15 mL tube and incubate 5 min on ice. Mix 2x by inverting the tube gently.
3. Pre-wet a 70 µm strainer cap with 0.5 mL of HB over a 50 mL test tube. Strain the nuclei suspension from step 2.2 by tipping over the 15 mL tube gently into the cell strainer. Wash the cell strainer with 0.5 mL of HB.
4. Remove the cell strainer and centrifuge the test tube at 500 x g for 5 min at 4 °C, using a swing bucket centrifuge. Discard the supernatant.
   NOTE: Straining the homogenate will help reduce the debris, which is critical for the flow cytometry and the snRNA-seq downstream steps.
5. Resuspend the pellet gently in 4 mL of HB using a P1000 pipette. Incubate on ice for 5 min. Spin at 500 x g for 10 min at 4 °C. Discard the supernatant and resuspend the pellet in 3 mL of WM.
6. Pre-wet a 35 µm strainer cap over a 15 mL test tube with 0.5 mL of WM. Strain the nuclei suspension from step 2.5 through the cell strainer, gently pipetting 0.5 mL at a time using a P1000 pipette.
7. Wash the strainer cap with 0.5 mL of WM and place the tube on ice. Transfer the filtrate to a new 15 mL tube and centrifuge for 5 min and 4 °C at 500 x g. Discard the supernatant and resuspend the pellet in 3 mL of WM.
8. Spin at 500 x g for 5 min at 4 °C. Discard the supernatant and resuspend the pellet in 1 mL of WM with the mouse anti-NeuN, Alexa Fluor 488-conjugated antibody (anti-NeuN-AF488, 1:32,000) and 1 µg/mL DAPI. Incubate for 45 min on ice in the dark.
   NOTE: To optimize the immunostaining of isolated nuclei, it is recommended to titrate the antibody to determine the optimal dilution for flow cytometry analysis and sorting. Then, run adequate controls to confirm that the staining conditions are optimal. For instance, with the conjugated anti-NeuN-AF488 antibody, a negative control (i.e., no addition of the antibody, Supplemental Figure 1A) and a positive control (i.e., staining with the antibody, Supplemental Figure 1B) were run to assess segregation of unstained and stained populations. When starting to work with a AF488-conjugated antibody, running an AF488-conjugated isotype control is recommended to assess specificity. If a non-conjugated antibody is used, additional control such as adding a secondary antibody only to a nuclei preparation might be necessary to assess unspecific binding of the secondary antibody.

3. Fluorescence activated nuclei sorting (FANS) to exclude neuronal populations (Timing: 45 min)

1. Transfer the immuno-stained nuclei suspension to a 5 mL test tube and keep it on ice until the start of the flow cytometry procedure.
   NOTE: If working with bigger pieces of tissue than two mouse DG, further dilution with WM buffer might be required to avoid clogging the FACS if nuclei density in the solution becomes high.
2. Vortex the samples for 3 s at low speed before placing the tubes into the FACS instrument (see Table of Materials).
   NOTE: (FACS setup) Sorting machines need to be aligned at the start of the procedure with calibration particles following the manufacturer's recommendations. The drop delay was calibrated with beads or microspheres (see Table of Materials) according to the FACS model. Samples were sorted at 4 °C on purity mode. To reduce the collection volume, the nuclei were sorted through a 70 µm nozzle at the recommended pressure for the flow cytometer. Nuclei were sorted into 1.5 mL low binding tubes (see Table of Materials) containing 50 µL of WM. All collection tubes were coated in PBS + 5% BSA at 4 °C overnight to reduce the risk of nuclei sticking to the walls of the tube.
3. To acquire the data from a sample of the stained nuclei suspension, set the gates in DAPI-height and the DAPI-area in order to exclude the cell debris and the aggregated nuclei (Figure 2A). Furthermore, separate single nuclei from any remaining DAPI-stained aggregates or cell debris by setting the gates in the log Side scatter (SSC)-area and the log Forward scatter (FCS)-area (Figure 2B).
4. Set the gates for the anti-NeuN-AF488 and the FSC-area, to isolate the NeuN-AF488-negative population, as shown in Figure 2C.
5. After analysis, using the gating strategy described above, sort the NeuN-AF488-negative population in a 1.5 mL collection tube filled with 50 µL of WM.
   NOTE: Following the gating strategy described above and the dissection procedure to isolate the DG from the brain of an adult mouse, the NeuN-AF488-negative population is expected to represent ~14% of the single nuclei.

Figure 2: Isolation and transcriptomic profiling of non-neuronal cell populations from the DG. (A–C) Gating strategy to isolate NeuN-AF488 negative single nuclei and exclude cell debris. (A) FANS dot plot of a representative sample of isolated nuclei, depicting the gate setting for the selection of DAPI+ nuclei and exclusion of cell debris and aggregates. (B) Further selection of relevant single nuclei using FSC-area and SSC-area. (C) The gates for NeuN-AF488 to exclude the positive population and sort for the negative single nuclei. (D) Micrograph of a good single nuclei suspension with minimum amount of debris and higher proportion of good quality nuclei (round shape, black arrow) compared to bad quality nuclei (white arrow). Scale bars = 50 µm, 10 µm (inset). (E,F) Analysis of snRNA-seq data and profiling of the distinct cell populations isolated from the DG of 22-month-old C57BL/6J male mice. Uniform Manifold Approximation and Projection for Dimension Reduction (UMAP) plots of single nuclei profiles from the (E) non-FACS-sorted cells and (F) NeuN-negative FACS-sorted cells, colored by cell type. (G) Pie charts comparing the frequencies of identified cell types in both samples. (H) Respective metrics for the sequenced samples: number of nuclei, median number of genes, and transcripts per nucleus. (I) Violin plots showing the distribution of the number of genes and transcripts detected for each cell type in both the samples. Astr. = astrocytes, Olig. = oligodendrocytes, Vasc. = vascular cells, CRCs = Cajal-Retzius cells, Neur. = neurons, Imm. = immune cells, OPCs = oligodendrocytes precursor cells. Please click here to view a larger version of this figure.

4. Preparation of the single nuclei suspension to perform single nuclei RNA sequencing (Timing: 30 min)

1. After sorting, add 1 mL of PBS containing 1% BSA to the collection tube in order to collect droplets on the wall of the tube and spin at 500 x g for 5 min at 4 °C. Discard the supernatant, leaving 50 µL.
   NOTE: Handle the centrifugated nuclei with care as it may be difficult to observe any pellet at the bottom of the tube. Using a swing-bucket centrifuge will help discard the supernatant without disrupting the pellet.
2. Pipet gently to resuspend centrifugated nuclei. Add 5 µL of the nuclei suspension to 5 µL of Trypan blue in a 0.5 mL microtube.
   CAUTION: Handle Trypan blue with care as it is hazardous for health, can cause cancer, and is suspected of damaging fertility or the unborn child. Wear protective gloves, clothing, and eye and face protection. Do not handle until all safety precautions have been read and understood.
3. Measure concentration and assess viability of the single cell suspension using a hemocytometer or an automated cell counter (see Table of Materials). Perform library preparation and sequencing of the nuclei as detailed in step 5.
   NOTE: Samples that were deemed to be of good quality for sequencing showed round and regular nuclei shape under the microscope with no cell debris (Figure 2D). The presence of a halo around the nuclear membrane or the aggregation of multiple nuclei together are signs of damaged nuclei and such cell suspensions should not be considered for snRNA-seq (Figure 2D). The measured concentration of nuclei was within the range of 300-700 nuclei/µL.

5. Library preparation and sequencing

NOTE: Description of the following steps is based on the in-house sequencing platform used in this study (see Table of Materials). Therefore, some settings might differ when using a different platform. Here, only the key steps are described and each parameter should be determined following guidance and protocols from the chosen manufacturer albeit with optimization before first use. It is critical to ensure that preparation of libraries is performed as quickly as possible after concentrating sorted nuclei suspensions to avoid RNA degradation and ensure optimal quality of the sequencing.

1. Load between 7,000 and 10,000 nuclei into a microfluidics Single Cell Chip.
2. Partition loaded nuclei in nanoliter scale droplets using the controller provided and the reagents from the chosen supplier. Lyse nuclei within each droplet and reverse transcribe RNA.
   NOTE: Within a droplet all the resulting cDNA shared the same cell barcode.
3. Prepare libraries for snRNA-seq following guidelines from the chosen supplier and ensuring compatibility with sequencing platform. Check the quality and concentration of final libraries using electrophoresis, fluorometry, or qPCR-based methods and, if applicable, pool them equimolarly prior to sequencing.
4. Denature pooled 3ʹ gene expression libraries and dilute as per the manufacturer's recommendation.
5. Perform paired-end, single or dual indexing sequencing on a next generation sequencing platform with sequencing depth of 50,000 read pairs per cell.