Apr 02, 2025
snMultiome-seq for Projection-TAGs
- 1Washington University in St. Louis
- Projection-TAGs
Protocol Citation: Lite Yang, Vijay Samineni 2025. snMultiome-seq for Projection-TAGs. protocols.io https://dx.doi.org/10.17504/protocols.io.yxmvm9dm5l3p/v1
Manuscript citation:
Yang L, Liu F, Hahm H, Okuda T… MR, Samineni VK. Projection-TAGs enable multiplex projection tracing and multi-modal profiling of projection neurons. bioRxiv [Preprint]. 2024 Apr 28:2024.04.24.590975.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: March 11, 2025
Last Modified: April 02, 2025
Protocol Integer ID: 124212
Keywords: High-throughput neuroanatomical tools, Multiplex projection tracing, Single-cell multiomic profiling, Projecting neurons, RNA barcodes, Projection-TAGs
Funders Acknowledgements:
National Institute of Diabetes and Digestive and Kidney Diseases
Grant ID: 5R01DK128475-04
Abstract
This is a master protocol includes the complete process for multiplex projection tracing using Projection-TAGs in snMultiome-seq assay. This master protocol includes the following procedures:
1. Nuclear extraction from fresh frozen tissues.
2. Isolation of Projection-TAG+nuclei using FACS.
3. snMultiome-seq library preparation, sequencing, and data processing.
For more Projection-TAGs protocols, please visit: https://www.protocols.io/workspaces/projection-tags
Materials
Equipment and Materials
- Bench-top centrifuge
- Swing-bucket rotor ultracentrifuge (Beckman, L-70)
- Swing-bucket rotor (Beckman, SW 28)
- Ultracentrifuge tube (4 ml thick-walled polycarbonate tube, Beckman Coulter 355645)
- Ultracentrifuge rotor adaptor (13mm Diameter Delrin Tube Adapter, Beckman Coulter 303392)
- 2 ml Dounce Tissue Grinder (Sigma-Aldrich, D8938-1SET)
- 2 ml DNA LoBind tube (Eppendorf, 022431048)
- 1.5 ml DNA LoBind tube (Eppendorf, 022431021)
- 5 ml FACS tube (Falcon, 352063)
- CellTrics filters (50 um, Sysmex, 04-004-2327)
- Pipettes and pipette tips
- Hemocytometer (Incyto, DHC-N01-5)
Reagents:
- Sucrose (Fisher scientific, AAJ6514836)
- PBS (10X, Fisher scientific, AAJ75889AE)
- Nuclease-free Water (Invitrogen, 10977023)
- Tris-HCl (pH 8.0, Invitrogen, AM9855G)
- NaCl (5 M, Invitrogen, AM9760G)
- KCl (2 M, Invitrogen, AM9640G)
- MgCl2 (1 M, Invitrogen, AM9530G)
- NP40 (10%, Thermo Scientific, 28324)
- Actinomycin D (2 mg/mL, Sigma-Aldrich, SBR00013)
- OptiPrep Density Gradient Medium (Sigma-Aldrich, D1556-250ML)
- BSA (Bovine Serum Albumin, 30%, Sigma-Aldrich, 126625-50ML)
- Protector RNase Inhibitor (Sigma-Aldrich, 3335402001)
- 7-AAD (1 mg/ml, Sigma-Aldrich, SML1633-1ML)
Before start
Please carefully read the critical steps in the protocol as they are specifically optimized for Projection-TAG experiment.
Using a bench-top centrifuge with swing-bucket rotor is recommended for improving nuclei yield.
Nuclear extraction
Nuclear extraction
2h 40m
2h 40m
Preparation
30m
Wipe clean the working area with 70% ethanol and RNase Zap.
Clean douncers with 70% ethanol, and rinse them with nuclease-free water for a total of 2X. Place them in clean 50 ml conical tubes and cover with Kimwipe.
Place SW28 rotor into the ultracentrifuge and pre-chill the bench-top centrifuge to 4C.
Note: using a centrifuge with swinging bucket rotor may improve nuclear extraction efficiency.
Prepare buffers and store them on ice.
Note: PBS, NIM, and Diluent can be prepared in advance and stored at 4C for up to 3 months. Filter NIM with 0.2 um filter.
| PBS | Stock | Final | 50 ml | |
| Nuclease-free Water | 45 ml | |||
| PBS | 10X | 1X | 5 ml |
| NIM (Nuclear isolation media) | Stock | Final | 100 ml | |
| Nuclease-free Water | 47.25 ml | |||
| Sucrose | 0.5 M | 0.25 M | 50 ml | |
| KCl | 2 M | 25 mM | 1250 ul | |
| MgCl2 | 1 M | 5 mM | 500 ul | |
| Tris-HCl, pH 8.0 | 1 M | 10 mM | 1000 ul |
| Diluent | Stock | Final | 50 ml | |
| Nuclease-free Water | 41.75 ml | |||
| KCl | 2 M | 150 mM | 3.75 ml | |
| MgCl2 | 1 M | 30 mM | 1.5 ml | |
| Tris-HCl, pH 8.0 | 1 M | 60 mM | 3 ml |
| A | B | C | D | E | F | |
| Homogenization Buffer | Stock | Final | 1100 ul per sample | X2 | X4 | |
| NIM | 1060 ul | 2120 | 4240 | |||
| BSA | 30% | 1% | 37 ul | 74 | 148 | |
| RNase inhibitor | 40 U/ul | 0.08 U/ul | 2.2 ul | 4.4 | 8.8 | |
| Actinomycin D | 2 mg/ml | 5 ug/ml | 2.75 ul | 5.5 | 11 |
| Resuspension Buffer | Stock | Final | 2000 ul per sample | X2 | X4 | |
| PBS | 1X | 1X | 1930 ul | 3860 | 7720 | |
| BSA | 30% | 1% | 66 ul | 132 | 264 | |
| RNase inhibitor | 40 U/ul | 0.08 U/ul | 4 ul | 8 | 16 |
| 50% iodixanol | 3 ml per sample | X2 | X4 | |
| Opti-prep | 2500 ul | 5000 | 10000 | |
| Diluent | 500 ul | 1000 | 2000 |
| 40% iodixanol | 1100 ul per sample | X2 | X4 | |
| 50% iodixanol | 880 ul | 1760 | 3520 | |
| NIM | 220 ul | 440 | 880 | |
| 30% BSA | 37 ul | 74 | 148 | |
| RNase inhibitor | 2.2 ul | 4.4 | 8.8 |
| 30% iodixanol | 1100 ul per sample | X2 | X4 | |
| 50% iodixanol | 660 ul | 1320 | 2640 | |
| NIM | 440 ul | 880 | 1760 | |
| 30% BSA | 37 ul | 74 | 148 | |
| RNase inhibitor | 2.2 ul | 4.4 | 8.8 |
Gather samples and store them on dry ice.
Prepare iodixanol gradient. Pre-indent ice with tubes in advance to avoid disrupting layers. Layer in ultracentrifuge tube 1 ml 40% at bottom of tube, and then slowly add on top of it, 1 ml 30% iodixanol solutions carefully. Slowly pipette just above 40% layer against the wall allowing it to form two distinct layers. Leave this on ice while you prepare the sample.
Homogenization
10m
Take one sample out from dry ice and thaw on wet ice for 1 min.
Quick spin to collect the tissue at the bottom of the tube.
Add 1 ml of Homogenization Buffer and 10 ul of 10% NP40.
Using a P1000 pipette, transfer samples and buffer to a pre-chilled douncer.
Insert the loose pestle (“A”) into the homogenizer and perform 15 strokes. Perform additional 15 strokes with the tight pestle (“B”).
Optional: if big chunks are still visible, remove the pestle and let the douncer sit on ice for 1 min allowing chunks to precipitate. Perform additional 5 strokes with the tight pestle (“B”). At the end of each stroke, rotate the pestle back and forth to further grind the sample.
Filter the homogenate directly with a 50 µm Sysmex filter into a 2 ml centrifuge tube.
Note: If there is abundant debris, filtration can be aided by taking your thumb and covering the top of the filter opening. This will create positive pressure that pushes the filtrate through the filter.
Bring the sample volume up to 1 ml using Resuspension Buffer.
Vortex 50% iodixanol. Add 1 ml of freshly-vortexed 50% iodixanol to the homogenate to make 25% iodixanol.
Cap the tube and keep on ice. Repeat Step 3 for additional samples.
Gradient centrifugation
1h
Invert sample tubes 10X to mix up nuclei resuspensions in 25% iodixanol.
Using a P1000 pipette, slowly pipette 2 ml of nuclear resuspensions on top of the 30% layer in the ultracentrifuge tube. Slowly pipette just above 30% layer against the wall allowing it to form two distinct layers.
Adjust volume of samples with nuclease-free water to match same level.
Centrifuge at 10,000 g for 18 min (8,900 rpm with SW28 rotor). Set the temperature to 4C.
When spin is finished, remove 1.5 ml of 25% iodixanol layer in order to get better access to the interlayer. Aspirate out the buffer from the surface to remove cell debris.
Stab a P200 pipette tip to the interface of 30% and 40% iodixanol. While hovering the pipette tip over the interface and moving around the surface, aspirate 200 ul of interlayer and transfer to a 1.5 ml centrifuge tube.
Note: Dots of nuclei may be visible interface between 30% iodixanol and 40% iodixanol layers.
Repeat step 3.6 for a total of 3 times to collect 600 ul from the interlayer.
Dilute iodixanol with 600 ul of Resuspension Buffer + 6 ul of 7-AAD ready-made solution (5 ng/ul final concentration). Pipet mix 10X.
Centrifuge the tubes with the bench-top centrifuge at 500 g for 10 min at 4°C.
Note: If you start with a small amount of tissues, the pellet may not be visible after centrifugation. Therefore, it’s important to note the position the tubes in the centrifuge so that you know where the pellet is supposed to be after centrifugation.
Gently remove one tube at a time from the centrifuge. Aspirate the supernatant by inverting the tube. Leave behind ~50 ul of supernatant as not to disturb nuclei pellet.
Resuspend the pellets in up to 500 ul of Resuspension Buffer. Pipet mix 10X.
Centrifuge the tubes with the bench-top centrifuge at 500 g for 5 min at 4°C to spin down excessive supernatant.
Using a P200 pipette, carefully remove the supernatant. Leave behind ~10 ul of supernatant as not to disturb nuclei pellet.
Optional: Load 10 ul on a hemocytometer. Inspect on an epifluorescence microscope with a 7-AAD filter.
FACS
FACS
2h 40m
2h 40m
Note: While not strictly necessary, FACS is highly recommended to enrich for labeled nuclei and thus improve the detection efficiency of Projection-TAGs in snMultiome-seq data.
Transfer the nuclear resuspensions to 5 ml FACS tubes.
Prepare collection tubes for each of the samples by adding 100 ul of Resuspension Buffer into a 1.5 ml tube.
Perform FACS using the 100 μm nozzle.
To select for single nuclei, first gate the FACS events on size, linearity, doublet, and 7-AAD signal.
To select for Projection-TAG+ nuclei, set up a secondary gate on GFP signal.
For Projection-TAG+ nuclei samples, sort and collect 7-AAD+ and GFP+ events in a 1.5 ml tube. For other nuclei samples, sort and collect 7-AAD+ and GFP- events in a 1.5 ml tube.
Note: Collecting at least 100k events per sample is recommended to target 10k cell recovery for snMultiome-seq.
snMultiome-seq library preparation and sequencing
snMultiome-seq library preparation and sequencing
Process the nuclei samples and prepare snMultome-seq library according to the user manual of 10X Genomics Chromium Single Cell Multiome ATAC + Gene Expression.
To ensure an optimal recovery of Projection-TAG UMIs, sequence the snRNA-seq library targeting at least 100k reads/nuclei. Sequence the snATAC-seq library according to the user manual.
snMultiome-seq data processing using cellranger-arc
snMultiome-seq data processing using cellranger-arc
Fetch the sequence of Projection-TAGs from https://github.com/Samineni-Lab/Projection-TAGs/tree/main/Map_reads.
Note: example cellranger-arc codes are also available on GitHub.
Generate a custom mouse reference genome containing the Projection-TAG features following the instructions given by 10X Genomics: https://www.10xgenomics.com/support/software/cell-ranger-arc/latest/analysis/inputs/mkref.
Run cellranger-arc by mapping the sequencing reads to the custom mouse reference genome: https://www.10xgenomics.com/support/software/cell-ranger-arc/latest/analysis/running-pipelines/single-library-analysis.
Quality control suggestions for Projection-TAG experiments
Quality control suggestions for Projection-TAG experiments
Ambient RNA contamination may lead to false detection of Projection-TAGs. False detection rate (FDR) should be determined by examining the detection rate of Projection-TAGs in cell populations that do not project to the targets examined in the experiment (e.g. non-neuronal cells).
Off-target AAV labeling may lead to non-specific labeling. To make sure the Projection-TAGs are delivered to the expected target region, tissue sections containing the injection target regions should be collected and the GFP signal around the target regions should be assessed.