Multiplexed snRNA-seq from frozen human brain samples

Check if the Rotor SW32Ti rotor is in the cold room

Clean bench and pipettes with RNAse Zap.

Add RNAse inhibitor (Sigma cat.# 3335402001 ) to LB and WRB (0.2U/ul).

Put lysis buffer and sucrose solutions on ice.

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

Dounce tissue on ice with 10 strokes or until no chunks of tissue are visible.On ice

Transfer homogenized tissue in lysis buffer into a labeled thick wall ultracentrifuge tube on ice (Beckman Coulter; 355631).On ice

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

When you are done with all samples weigh them and bring to the same weight by adding Lysis buffer.

Load the samples to SW32Ti rotor (needs to be swing bucket). If using less than 6 samples still balance with empty buckets.107163rcf,4°C

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.

Meanwhile, transfer materials to the tissue culture room. Prepare eppendorf tubes with 10ul of DAPI for each sample.

Add 800ul of WRB (for a total of 1ml of WRB)and resuspend cells.On ice

Filter twice using Miltenyi Pre-separation filters (30um). (130-041-407)

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

Thaw Cell Multiplexing Oligo at room temperature. Vortex for 5 sec and centrifuge for 5 sec.

Centrifuge nuclei using a swing bucket rotor 500rcf,4°C

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

Add 1.9 mL of cold WRB supplemented with RNAse inhibitor. Gently pipette mix

Centrifuge cells using a swing bucket rotor 500rcf,4°C

Remove supernatant and add 2ml of cold WRB.

Centrifuge cells using a swing bucket rotor 500rcf,4°C

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.

Pool samples together and count the total number of cells using a hemocytometer.

Load 49,500 cells/10x well to aim for 30k cells

After sequencing your libraries, align your reads with CellRanger and make sure to enable the option to generate .BAM files

Familiarize yourself with popscle/freemuxlet inputs, outputs and vignetes on its Github page:

https://github.com/statgen/popscle

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.

After running, identify the droplet barcodes that were not assigned as singlets and remove these cells from your analysis

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.