title: GSM8335467: 113_ND_BCR; Homo sapiens; OTHER
library_construction_protocol: Cells were thawed in a 37 C waterbath, washed twice with 10 mL 1X PBS, and kept on ice for cell staining. Cells were stained with LIVE/DEADTM Fixable Near-IR Dead Cell Stain Kit at a 1:1500 dilution (ThermoFisher, Cat#L34975) in FACS buffer (1% BSA in 1X PBS) for 15 minutes on ice in the dark. Then, 2 ug of INS tetramer, 1 ug of IA2 and TET tetramers, and 0.75 ug of GAD and Empty tetramers in addition to 1:200 anti-human CD19-BV421 (BioLegend, Cat#363017) and 1:100 anti-human CD3-FITC (BioLegend, Cat#317305) were added to ~30e6 PBMCs in 1 mL total volume. Cells were stained on ice for 20 minutes in the dark. Finally, cells were washed twice in FACS buffer (400 x g for 5 minutes) prior to analysis on the cell sorter. After tetramer and antibody staining, cells were immediately brought to the CU Cancer Center Flow Cytometry Core. PE-binding B cells were sorted as Live/singlet/lymphocyte/CD19+/CD3-/PE+ cells into 100 uL of FACS buffer. Up to 20,000 B cells were collected, and in some instances, PE-negative binding B cells were also sorted to increase the overall yield of B cells to be captured. After the cells were sorted, they were stained with 1 uL each of the following TotalSeqC surface antibodies: anti-human IgM (BioLegend, Cat#314547), anti-human IgD (BioLegend, Cat#348245), anti-human CD27 (BioLegend, Cat#302853), anti-human CD21 (BioLegend, Cat#354923), and anti-human CXCR5 (BioLegend, Cat#356939). Cells were stained for 15 minutes on ice in the dark, washed once  (400 x g for 5 minutes), and then loaded onto the 10X Genomics system for cell capture at the CU Genomics Core. The 10X Genomics Chromium system was used for 5' mRNA capture with chip K according to user guide CG000330_ChromiumNextGEMSingleCell5-v2_CellSurfaceProtein_UserGuide_RevF.pdf.  The deviations from this protocol included: 1.) Overloading the chip with up to 20,000 cells per well depending on the sample, and 2.) Using the Feature Barcode Kit Primer 4 (Cat# 2000277) to detect and amplify TotalSeq C barcodes. Otherwise, the protocol was followed as written to produce gene expression, BCR V(D)J, and Feature Barcode Cell Surface Protein libraries for sequencing analysis. Library quality and quantity was determined using Agilent High Sensitivity D1000 TapeStation Screentape (Cat# 5067-5584), Reagents (Cat# 5067-5585), and Qubit DNA Quantification Assay (Cat# Q32851).