Nuclei Isolation and FANS
Here, we provide a summary of the progress made so far regarding unfixed nuclei isolation from post-mortem human brain samples and fluorescence-activated nuclei sorting (FANS) protocols in our lab. We have evaluated and further optimised protocols from the Nott and Paul Matthews Labs to better suit our experimental needs. Our research focuses on both the epigenome and transcriptome, which influences our choice of protocols to ensure compatibility with both types of analyses. The following sections detail the differences between the initial and current protocols, the reasons for the change,as well as the additional optimisations implemented so far.
- Sample type: Originally designed for frozen resected human tissue. Used for post-mortem tissue in our lab
- Sample preparation: Manual cutting of tissue in the hood, which can lead to inconsistencies and increased tissue loss
- Volumes and equipment: Utilises 15 mL Falcon tubes and a 7 mL douncer, increasing the risk of nuclei loss and requiring more reagents. The protocol uses a large centrifuge with a swinging bucket to ensure nuclei concentrate at the bottom of the tubes
- Density gradient: No sucrose density gradient used, which may be less effective for post-mortem tissues that are generally less clean, leading to increased debris and background autofluorescence
- Antibodies: NeuN-488 (MAB377X) and Olig2-647 (AB225100) pre-conjugated antibodies with overnight incubation at 4°C, leading to RNA degradation
Reference: Reference paper for Nott Lab Protocol
Full protocol: Alexi Nott Lab protocol.docx
- Suitable for cleaner, resected tissue samples.
- Post-mortem tissues, which often require density gradients to manage debris and improve purity
- Experiments needing preservation of RNA integrity for downstream assays due to the extended incubation times
- Situations where precise tissue amounts are critical for downstream experimental analysis
- Sample type: Frozen post-mortem human tissue
- Sample preparation: Uses a cryostat to cut samples into 80 µm sections, reducing stress and improving consistency
- Volumes and equipment: Employs smaller volumes and equipment, including a 2 mL douncer and 1.5 mL low-bind Eppendorf tubes, reducing reagent use and nuclei loss. The small centrifuge used has a fixed angle, causing nuclei to stick to the tube walls; this is somewhat mitigated by coating the tubes with BSA prior to the start of the experiment
- Density gradient: Includes a sucrose density gradient to manage post-mortem tissues effectively, helping to reduce autofluorescence
- Antibodies: NeuN (MAB377) and Sox10 (AF2864) antibodies (unconjugated) with a 1-hour incubation. Secondary antibodies (488 anti-goat and 647 anti-mouse) have a 30-minute incubation time, reducing total antibody incubation to 1 hour and 30 minutes. This approach helps preserve RNA integrity for downstream assays
- scRNA-seq Compatibility: Optimised for scRNA-seq and used to prepare nuclei for the 10X multiome sequencing (ATAC + gene expression) assay
Reference: Reference paper for Matthews Lab Protocol
Full Protocol: Paul Matthews Lab protocol.docx
- Ideal for post-mortem tissues, as it includes a density gradient that helps manage debris and reduces autofluorescence
- Uses cryostat-cut samples, which reduces stress and allows consistency
- Optimised for scRNA-seq, preserving RNA integrity
- Samples requiring very large volumes where a larger centrifuge might be preferred
- Low-retention tips: Utilised low-retention tips to reduce nuclei loss, minimising nuclei sticking to pipette tips
- Centrifuge adaptation: Combined the use of a large centrifuge with a swinging bucket for better nuclei pelleting (from the Nott protocol) with smaller volumes (1.5 mL low-bind Eppendorf tubes) to reduce reagent use (from the Matthews protocol), integrating the advantages of both approaches
- Filtering steps: Reduced FACS cap filtering steps from three to one before sorting to improve efficiency and reduce sample loss. Changed the 70 µm strainer midway through the density gradient filtering step to prevent blockage and sample loss
- Ultracentrifugation step: Reduced duration from 40 to 30 minutes.
To further optimise the protocol, we tested various staining strategies:
- Initial strategy: Used the staining strategy suggested by the Paul Matthews Lab, with unconjugated NeuN and Sox10 antibodies. After obtaining clear populations, we tested additional optimisations described above to enhance nuclei yield
- Alternative antibodies: Tested NeuN-488, Sox10-NL577, and IRF8-APC according to Jon Mill's. However, our FACS machine (BRC Aria II) lacks the necessary laser to detect NL577
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Microglia Sorting:
- Tested two rabbit Ikaros antibodies (product codes: A303-516A and PA5-95958), stained with AF647. Both antibodies showed significant non-specific binding, potentially due to high concentrations
- Tested NeuN-488, Olig2-647, and IRF8-PE (clone: E6J8Q) with a reduced incubation time of 1 hour. This combination did not yield an IRF8+ population. All antibodies are pre-conjugated
- Tested NeuN-488, Olig2-647, and IRF8-PE (clone: V3GYWCH) with the standard incubation time of 1 hour 30 minutes. This combination successfully identified an IRF8+ population with good counts. All antibodies are pre-conjugated. See the table below:
Full tested antibody table: Antibodies-nuclei staining.xlsx
Current gating strategy as per Jon Mill's protocol.
To adapt the protocol for proteomics purposes we performed two experiments:
- Experiment 1: Lowered final BSA concentration from 1% to 0.1% before staining, then removed BSA and topped up with PBS. Observed nuclear clumping and low nuclei yield
- Experiment 2: Added 250 mM sucrose to the buffer before and after staining, in addition to lowering and removing BSA as described in Experiment 1. No clumping was observed, but nuclei counts were even lower. See the table below:
To address autofluorescence issues, we tested the use of True Black dye:
- We split the sample (100 mg each) into standard and True Black conditions (1:20 dilution in 70% ethanol). Reduced antibody incubation to 1 hour
- Results: Identified four populations: NeuN+, Olig2+, IRF8+, and triple negative. True Black seems to reduce background signal but also decreased overall staining intensity. The NeuN+ population decreased, and the IRF8+ population was less well-separated compared to previous experiments