TITLE: Multiplexed snRNA-seq from  frozen human brain samples
AUTHORS: Marcos Nascimento

[DESCRIPTION]
This is a protocol to save your experiment and successfully demultiplex nuclei using CellPlex, the cholesterol-modified-oligo (CMO) barcodes from 10x Genomics on your nuclei from snap-frozen tissue.

Generally speaking, nuclei from snap-frozen tissue perform really poorly with CMOs.  This crucial information was not in their initial documentation when the product was launched, leading to costly losses.

However, if you are multiplexing samples from different human donors, there is a way of saving your data and still demultiplexing your samples quickly and reliably. By taking advantage of known SNPs in the human genome, it is possible to demultiplex samples originating from different donors using freemuxlet.

[BEFORE_START]
Important: If you are designing an experiment and planning to multiplex your nuclei from frozen tissue, you should avoid using Cellplex altogether. It's better to design your experiment using freemuxlet in combination with DNA sequencing or bulk RNAseq from each donor. If you already used CellPlex and your barcoding is terrible, this protocol is for you.

[STEPS]
SECTION: Preparation
1. Check if the Rotor SW32Ti rotor is at 4°C
SECTION: Preparation
2. Prepare your working area: Clean the workbench and pipettes with RNAse Zap.
SECTION: Preparation
3. Add RNAse inhibitor (Sigma cat.# 3335402001) to LB and WRB (0.2U/ul).
SECTION: Preparation
4. Put lysis buffer and sucrose solutions on ice.

SECTION: Nuclei Isolation
5. Use glass dounce homogenizer (Thomas Scientific; Catalog # 3431D76; size A). Put douncer on ice, pipette 1mL of lysis in the douncer. Transfer tissue either using spatula or P1000 pipette with cut tip and additional lysis buffer. Bring the total volume of lysis buffer in the douncer to 3mL.on ice 

SECTION: Nuclei Isolation
6. Dounce tissue on ice with 10 strokes or until no chunks of tissue are visible.on ice 

SECTION: Nuclei Isolation
7. Transfer homogenized tissue in lysis buffer into a labeled thick wall ultracentrifuge tube on ice (Beckman Coulter; 355631).on ice 

SECTION: Nuclei Isolation
8. Carefully pipette 9 mL of Sucrose solution to the bottom of the tube containing Lysis buffer. Be careful not to introduce bubbles. You should see two clearly separated phases: sucrose on the bottom and cloudy homogenate on top.on ice 

SECTION: Nuclei Isolation
9. When you are done with all samples weigh them and bring to the same weight by adding Lysis buffer.
SECTION: Nuclei Isolation
10. Load the samples to SW32Ti rotor (needs to be swing bucket). If using less than 6 samples still balance with empty buckets.107163 rcf, 150 min, 4 °C 

SECTION: Nuclei Isolation
11. After the spin, transfer samples on ice and carefully remove the supernatant using a P200 tip cut at an angle and vacuum. Make sure not to touch the bottom (stick to the wall and tilt the tube), but remove all the liquid. Carefully pipette 200uL of WRB on the bottom. Wait 20 min on ice.
SECTION: Nuclei Isolation
12. Meanwhile, transfer materials to the tissue culture room. Prepare eppendorf tubes with 10ul of DAPI for each sample.
SECTION: Nuclei Isolation
13. Add 800ul of WRB (for a total of 1ml of WRB)and resuspend cells.on ice 
SECTION: Nuclei Isolation
14. Filter twice using Miltenyi Pre-separation filters (30um). (130-041-407)
SECTION: Nuclei Isolation
15. Add 10ul of each sample to 10ul of DAPI. Count nuclei in each sample using a hemocytometer. 
You should have at least 105 nuclei/sample 
 

SECTION: CellPlex Barcoding
16. 
Thaw Cell Multiplexing Oligo at room temperature. Vortex for 5 sec and centrifuge for 5 sec.
SECTION: CellPlex Barcoding
17. Centrifuge nuclei using a swing bucket rotor 500 rcf, 10 min, 4 °C 
SECTION: CellPlex Barcoding
18. Remove supernatant
Add 100ul of Cell Multiplexing Oligo (room temperature) to the nuclei. Gently pipette mix 10-15x to resuspend. Incubate for 5 mins.
Store the remaining Cell Multiplexing Oligo at -20C
SECTION: CellPlex Barcoding
19. Add 1.9 mL of cold WRB supplemented with RNAse inhibitor. Gently pipette mix

SECTION: CellPlex Barcoding
20. Centrifuge cells using a swing bucket rotor 500 rcf, 10 min, 4 °C 
SECTION: CellPlex Barcoding
21. Remove supernatant and add 2ml of cold WRB. 
SECTION: CellPlex Barcoding
22. Centrifuge cells using a swing bucket rotor 500 rcf, 10 min, 4 °C 
SECTION: CellPlex Barcoding
23.   

SECTION: CellPlex Barcoding
24. Based on starting concentration and assuming a ~50% cell loss, add an appropriate volume of cold WRB to obtain a final concentration of ~1500 cells/ul.
SECTION: CellPlex Barcoding
25. Pool samples together and count the total number of cells using a hemocytometer.
Load 49,500 cells/10x well to aim for 30k cells  

SECTION: Demultiplexing with popscle/freemuxlet
26. After sequencing your libraries, align your reads with CellRanger and make sure to enable the option to generate .BAM files 
SECTION: Demultiplexing with popscle/freemuxlet
27. Familiarize yourself with popscle/freemuxlet inputs, outputs and vignetes on its Github page:

https://github.com/statgen/popscle
SECTION: Demultiplexing with popscle/freemuxlet
28. Use the BAM files as inputs to freemuxlet. A .VCF file (a reference file with the SNPs) is also needed as an input for freemuxlet and can be obtained from 1000 genomes, filtering for high variant confidence, Minor Allele Frequency (MAF 0.01) and exonic variants.
SECTION: Demultiplexing with popscle/freemuxlet
29. After running, identify the droplet barcodes that were not assigned as singlets and remove these cells from your analysis
SECTION: Demultiplexing with popscle/freemuxlet
30. In order to identify which donor identified in freemuxlet corresponds to each sample, you can simply check for the amount of each CMO in all cells of each donor. Despite CMO labeling being too weak to demultiplex samples by itself, you can use it to match donors identified on freemuxlet with each sample.