Facs aria 3 flow sorter

The MND (myeloproliferative sarcoma virus enhancer, negative control region deleted, dl587rev primer binding site substituted) promoter sequence was PCR amplified from PTRip-MND-GFP (a gift from Francoise Pflumio) and cloned into pZIP-hCMV-ZsGreen-Puro lentiviral vector (Transomics technologies Inc.) using ClaI and AgeI restriction sites to generate pZIP-MND-ZsGreen-Puro. Short hairpin RNAs (shRNAs) against B-cell lymphoma/leukemia 11A (BCL11A) gene were obtained from the Sherwood shRNA design algorithm [45 (link)]. The shRNA oligo was amplified and cloned into HpaI digested pZIP-MND-ZsGreen-Puro using the NEBuilder Hi-fi DNA assembly cloning kit (NEB Biolabs Inc., Ipswich, MA, USA) to generate the pZIP-MND-ZsGreen-Puro-shBCL11A plasmid. PBiEPC-1, PBiEPC-2, and CD34iEPC were transduced with the lentiviruses in the presence of 8 µg/mL polybrene (Sigma-Aldrich, St. Louis, MO, USA). After five days, the ZsGreen⁺ cells were sorted using a BD FACS Aria III flow sorter (BD Biosciences). BCL11A knockdown was analyzed by western blot and qPCR in the undifferentiated PBiEPC-1 cells. Fetal hemoglobin (HbF) and γ-globin levels were measured in the differentiated PBiEPC-1 using flow cytometry and HPLC, respectively.

Patient cells were harvested as described above for flow cytometry. Cryopreserved samples for 10x were rapidly thawed and washed with complete RPMI containing 50 U ml⁻¹ of benzonase (Merck Life Science Limited). Cells were then stained with CAR anti-idiotype, followed by goat anti-rat IgG PE antibody and antibodies to CD3 APC (UCHT1, BioLegend, 1/20) and CD45 FITC (2D1, BioLegend, 1/20). DAPI was used to distinguish viable cells. CAR-T cells were isolated as CD45⁺CD3⁺CAR⁺ events in a live singlet leukocyte forward-scatter (FSC)/side-scatter (SSC) gate using a BD FACSAria III flow sorter. The flow cytometry gating strategy for CAR sorting can be found in Extended Data Fig. 10. CAR and non-CAR populations were sorted simultaneously and then immediately used downstream for the 10x workflow. Flow-sorted cells (CAR and non-CAR) were loaded according to the standard protocol of the Chromium Single Cell 5′ Kit (v2 chemistry). A TCR single-cell library was subsequently prepared from the same cells with the Chromium Single Cell V(D)J Enrichment Kit. The 5′ gene expression library and the TCR single-cell library were pooled with a molar ratio 10:1 for sequencing on Illumina NovaSeq S4 with 28 × 90 bp, aiming for an average of 300,000 reads per cell for the 5′ gene expression library and 30,000 reads per cell for the TCR single-cell library.