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.

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.本数据集包含来自健康骨髓（Bone Marrow, BM）与B细胞急性淋巴细胞白血病（B-cell Acute Lymphoblastic Leukemia, B-ALL）患者来源异种移植（Patient-Derived Xenograft, PDX）样本的单细胞RNA测序与抗体标签测序数据，上述样本取自抗CD19嵌合抗原受体T细胞（CD19-directed CAR T cells）治疗前及治疗后，受试者编号为CR2、CR4、CR6、Neg2、Neg5、Neg11。 所有样本经复苏后于37℃静置30分钟。随后将细胞通过35μm孔径细胞筛过滤，并以350g离心5分钟。将细胞重悬于1ml染色缓冲液（Stain Buffer，BD生物科学（BD Biosciences）），并按照制造商说明书使用人Fc封闭剂（Human Fc Block，BD生物科学）进行封闭。为富集Lin⁻/B⁺细胞组分，将样本与生物素偶联抗体（补充表3）共孵育30分钟。用染色缓冲液洗涤细胞后，按照制造商推荐浓度加入BD链霉亲和素磁珠复合物（BD Streptavidin Particles Plus，BD生物科学），于室温（Room Temperature, RT）共孵育30分钟。将标记磁珠的细胞置于磁力架上静置6分钟，将上清转移至新离心管，再次放回磁力架进行一轮磁珠结合细胞的耗竭操作并回收上清，随后以350g离心5分钟沉淀上清中的细胞。 将富集得到的Lin⁻/B⁺细胞组分重悬于180μl染色缓冲液，其中添加针对CD19、CD20、CD24、CD34、CD38、CD45、CD127及IgM的BD AbSeq寡核苷酸偶联抗体混合液（BD生物科学，补充表3）。随后使用人单细胞样本多重化试剂盒（Human Single Cell Sample Multiplexing kit，BD生物科学）对每个样本进行标记，于室温孵育30分钟。用染色缓冲液洗涤细胞两次，并重悬于样本缓冲液（BD生物科学）中。使用Countess II细胞计数器（Countess II Cell Counter，赛默飞世尔科技（Thermo Fisher Scientific））对细胞进行计数。将总计50,000个细胞（每个样本12,500个，最多可纳入4个样本）进行混合，使用BD Rhapsody Express单细胞分析系统（BD Rhapsody Express Single-Cell Analysis System，BD生物科学）在BD Rhapsody微流控芯片中分离单细胞。每个微流控芯片内均设置一份健康骨髓对照样本，以控制批次效应。本研究共使用3个微流控芯片。针对每个芯片，均按照制造商说明书，使用全转录组分析（Whole Transcriptome Analysis, WTA）扩增试剂盒（BD生物科学）制备全转录组、抗体标签及样本标签测序文库。同一芯片的文库使用相同的Illumina测序接头进行索引。将最终构建的文库混合后，于美国加利福尼亚州福斯特城的MedGenome公司使用NovaSeq 6000测序仪（NovaSeq 6000 sequencer，Illumina）进行双端100碱基对（base pair, bp）测序。 Fastq文件通过七桥平台（Seven Bridges platform，https://www.sevenbridges.com）上的Rhapsody分析流程（Rhapsody analysis pipeline，BD生物科学）进行处理，遵循制造商的推荐流程。使用bowtie2将测序reads比对至hg38参考基因组。本次上传的文件为以CSV格式存储的、经递归替换误差校正（Recursive Substitution Error Correction, RSEC）调整后的单细胞分子计数表达矩阵。