Single cell antigen receptor analysis reveals lymphocyte developmental origins
Single cell antigen receptor analysis reveals lymphocyte developmental origins
A vial of frozen PBMCs was acquired from Stemcell Technologies with informed consent (as stated by Stemcell Technologies) and approval from the Yorkshire & The Humber - Leeds East Research Ethics Committee (19/YH/0441). Frozen PBMCs were thawed in pre-warmed RF10 media, which was RPMI (Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; Gibco) and penicillin/streptomycin (Sigma-Aldrich). Cells were pelleted by centrifugation at 500g for 5 min and resuspended in RF10 media, and split between two 10 cm petri dishes. Control PBMCs were then incubated in a total of 10 ml RF10 media at 37°C for 2 hr, whereas treated PBMCs were incubated in RF10 supplemented with cycloheximide (Sigma-Aldrich; final concentration of 100 μg/ml). After incubation, control and treated PBMCs were washed with ice cold RF10 and resuspended in 2% FBS in phosphate buffered saline (PBS; Gibco). For treated PBMCs, both the washing and resuspension buffer contained 100 μg/ml cycloheximide.  Control and treated PBMCs were then loaded onto two separate channels of the Chromium chip from Chromium single cell V(D)J kit (10X Genomics 5′ v2) following the manufacturer's instructions before droplet encapsulation on the Chromium controller.  Single-cell cDNA synthesis was performed following the manufacturer's instructions.  Single-cell gene expression (GEX) and targeted BCR and αβTCR libraries were generated following the manufacturer's instructions.