CITE-seq data from Domizi, P et al. Nat Commun. 2025. IKAROS facilitates antigen escape in the face of CD19- and CD22-targeted therapies for B-cell malignancies.
Description
Single cell RNA and antibody tag sequencing data from healthy BM and B-ALL PDX samples from pre- or post- CD19-directed CAR T cells (Patient ID: CR2, CR4, CR6, Neg2, Neg5, Neg11). Samples were thawed and rested at 37 °C for 30 min. Then cells were filtered through cell strainer size 35 µm and centrifuged at 350g for 5 min. Cells were resuspended in 1 ml of Stain Buffer (BD Biosciences) and blocking was performed with Human Fc Block (BD Biosciences) following the manufacturer's instructions. To enrich for Lin−/ B+ fraction, samples were incubated with biotin-conjugated antibodies (Supplementary Table 3) for 30 min. Cells were washed with Stain Buffer and then incubated with BD Streptavidin Particles Plus (BD Biosciences) at the manufacturer's recommended concentration for 30 min at RT. Particle-labeled cells were placed in a magnetic holder for 6 min. The supernatant was transferred to a new tube and placed back in the magnetic holder for an additional round of depletion and supernatant recovery. Cells from the supernatant were then pelleted by centrifugation at 350g for 5 min. Lin-/ B+ fraction was resuspended in 180 µl of Stain Buffer supplemented with a mix of BD AbSeq oligo conjugated antibodies against CD19, CD20, CD24, CD34, CD38, CD45, CD127, and IgM (BD Biosciences, Supplementary Table 3). Then, each sample was labelled with the Human Single Cell Sample Multiplexing kit (BD Biosciences) and incubated at RT for 30 min. Cells were washed twice with Stain Buffer and resuspended in Sample Buffer (BD Biosciences). Cell number was counted with Countess II Cell Counter (Thermo Fisher Scientific). A total of 50,000 cells (12,500 cells per sample, up to 4 samples) were pooled together, and single cells were isolated in a BD Rhapsody cartridge using BD Rhapsody Express Single-Cell Analysis System (BD Biosciences). We included one healthy BM reference sample within each cartridge to control for batch effects. A total of 3 cartridges were used in this study. For each cartridge, we followed the manufacturer’s instructions to prepare whole transcriptomic, antibody tag, and sample tag libraries using the Whole Transcriptome Analysis (WTA) Amplification Kit (BD Biosciences). Libraries from the same cartridge were indexed with identical Illumina sequencing adapters. Final libraries were pooled together sequencing on a NovaSeq 6000 sequencer (Illumina) at MedGenome (Foster City, CA, USA) with paired-end 100 base pair (bp) reads. Fastq files were processed through the Rhapsody analysis pipeline (BD Biosciences) on the Seven Bridges platform (https://www.sevenbridges.com) following the manufacturer’s recommendations. Reads were mapped to the hg38 reference genome using bowtie2. Files deposited here contain expression matrices of recursive substation error correction (RSEC) adjusted molecule counts per cell in a CSV format.