Sep 13, 2024
Preparation of B cells for scRNAseq
- 1University of Pittsburgh
Protocol Citation: Nicholas Pease 2024. Preparation of B cells for scRNAseq. protocols.io https://dx.doi.org/10.17504/protocols.io.n92ld8e1nv5b/v1
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: September 13, 2024
Last Modified: September 13, 2024
Protocol Integer ID: 107559
Abstract
This protocol details activated B cell preparation and sample hashtagging for Chromium Single Cell Gene Expression (10x Genomics).
Materials
- EasySep‱ Dead Cell Removal (Annexin V) Kit (STEMCell Technologies, Cat. # 17899)
- TotalSeq‱-C0262 anti-human Hashtag 12 Antibody (Biolegend, Cat. # 394683)
- TotalSeq‱-C0263 anti-human Hashtag 13 Antibody (Biolegend, Cat. # 394683)
- TotalSeq‱-C0264 anti-human Hashtag 14 Antibody (Biolegend, Cat. # 394683)
- TotalSeq‱-C0265 anti-human Hashtag 15 Antibody (Biolegend, Cat. # 394683)
Dead cell removal
Dead cell removal
Pellet cells at 300xg for 10min
Resuspend in 100uL Dead Cell Removal Buffer and filter through 40uM cell strainer
Add 5uL of Annexin V Cocktail to sample
Add 5uL of Biotin Selection Cocktail to sample, mix and incubate at RT for 3min
Vortext RapidSpheres for 30 sec and add 10uL to each sample, flick to mix
Add 2.4mL of Dead Cell Removal buffer and mix again
Place tubes on magnet and incubate at RT for 10 min
Transfer supernatant to new 15mL tube
Aliquot
Sample hashtagging
Sample hashtagging
Resuspend samples in 100uL of wash buffer (PBS + 10% FBS)
Add 1uL of HTO antibodies
Incubate for 30 min on ice
Add 3.5mL of wash buffer to labeled cells, mix and pellet at 200xg for 10min at 4C
Remove supernatant with pipette, resuspend pellet with 3.5mL of wash buffer, and transfer to new tube
Pellet at 200xg for 10min at 4C
Remove supernatant with pipette and resuspend pellet with 3.5mL of wash buffer
Pellet at 200xg for 10min at 4C
Assuming 50% cell loss, resuspend pellet in wash buffer to obtain ~1,000 cells/uL
Count cells and adjust concentration, if needed
10x loading
10x loading
Mix hashtag samples at desired ratio and load 52,500 cells per well