CITE-seq. antibody staining protocol

December 2022

Whole bone crushed or use pelvic and hind limbs to spin down
Stain cells from each sample using antibody cocktails in 600µl staining media:
• APC, cKit, 1:200
• FITC, CD45.2, 1:200 (optional)
• PE-Cy7, Ter119, 1:500 (if LK)
• PE-Cy7, B220, 1:500 (if LK)
• PE-Cy7, CD3, 1:500 (if LK)
• PE-Cy7, Gr1, 1:500 (if LK)
• Biotin, Sca-1, 1:200 (if LSK)
Wash and spin down
Stain cells with anti-APC nanobeads: 1:20 in 600µl staining media
Wash and spin down
C-kit selection using column
Spin down, stain with DAPI, and filter for sort
Yield sort 250K cells of interest into 1.5ml Eppendorf tube with 100µl staining media
Spin down in bucket centrifuge and observe pellets
Make 30µl TotalSeq antibody cocktail per sample: 0.5µl of each antibody and adjust final volume with staining media

For example, if 4 genotypes and 2 biological replicates per genotype for LK cells:

Prepare Hashtag 1 master mix for 4 samples:

Prepare Hashtag 2 master mix for 4 samples:

Add ~28µl master mix to each tube of sorted cells and gently resuspend to mix
Incubate on ice for 30min
Add 1ml of staining media to wash
Spin down and remove supernatant
Add 1ml of staining media to wash again
Spin down and remove supernatant
Resuspend cells in 200µl of staining media with DAPI
Combine cells with Hashtags 1 and 2 from the same genotype, if applicable
Purity sort 100K cells into 300µl PBS + 0.01% RNase-free BSA in Eppendorf tube
Spin down and pipet out supernatant, leaving ~100µl for 1000 cells/µl
If there are plenty of cells, stain 10µl cells with 10µl Trypan blue to check recovered cell numbers and viability