title: GSM9241049: WT_B2; Mus musculus; RNA-Seq
library_construction_protocol: 9G4 antibody (purified rat anti-human IGHV4-34*01; IgM Biosciences) was conjugated to Alexa Fluor 647 (AF647) using an antibody labelling kit (Life Technologies), according to the manufacturer's instructions. Mouse bone marrow and spleen single cell suspensions were washed and kept in FACS buffer (PBS/5% FCS/0.02% sodium azide). All antibody incubation steps were completed on ice and in the dark, with centrifugations at 2000 rpm for 3 minutes at 4°C. Cells were incubated with anti-CD16/32 (ebioscience) Fc block for 15 minutes, washed twice incubated for 30-45 minutes, with a cocktail of flow cytometry antibodies (including 9G4) in PBS. After two washes, cells were fixed for 15 minutes in PBS/5% formalin (2% formaldehyde; Sigma) and resuspended in FACS buffer, for acquisition on the Symphony (BD) flow cytometer. A total of 27,000 and 28,000 peritoneal cavity B2 cells were bulk sorted into 1.5ml tubes containing sterile PBS/10% FCS from n=2 sex- and age-matched pre589-tg mice. A total of 10,000 cells were captured in the 5' V(D)J+GEX 10x reaction using the Chromium Single-Cell 5' Gel Beads and v2 Library kits (10X Genomics). Quality control of libraries was assessed with an Agilent BioAnalyzer High Sensitivity DNA chip, pooled, and quantified using qPCR (KAPA Library Quantification Kit). Denatured libraries were loaded onto an Illumina NovaSeq 6000 and sequenced using a 150-cycle High-Output Kit.