title: GSM8881334: Off-CAR-T chip; Homo sapiens; RNA-Seq
library_construction_protocol: The leukemia samples (designated as HD CAR, PD CAR, Mock, None) were engineered and cultured on-chip for 7 days as described above followed by 2-day treatment with CAR T cell, PD CAR T cell, Mock T cell or left untreated. Single‐cell suspensions of leukemia samples were prepared by off-chip recovery with nattokinase, pre-labeled with different anti-human hashtag antibodies (TotalSeq, BioLegend), i.e., Hashtag1-GTCAACTCTTTAGCG, Cat#394601; Hashtag2-TGATGGCCTATTGGG, Cat#394603, Hashtag3-TTCCGCCTCTCTTTG, Cat#394605, Hashtag4-AGTAAGTTCAGCGTA, Cat#394607, and mixed into one sample. Correspondingly, freshly thawed HD CAR T cell, PD CAR T cell, Mock T cell, and bone marrow mononuclear cells were pre-labeled with different anti-human hashtag antibodies, mixed into one sample, and used controls. The libraries were prepared using the Chromium Single Cell 3′ Reagent Kits (v3): Single Cell 3′ Library & Gel Bead Kit v3 (PN-1000075), Single Cell 3′ Chip Kit v3 (PN-1000073) and i7 Multiplex Kit (PN-120262) (10x Genomics) and following the Single Cell 3′ Reagent Kits (v3) User Guide (manual part no. CG000183 Rev B). Libraries were then run on an Illumina NovaSeq 6000 using 28bp read 1, 8bp i7 index, and 91bp read 2.  The raw sequencing results from the 10x Genomics platform were processed using CellRanger (version 3.1). In brief, CellRanger was used to generate a count matrix by aligning output reads, filtering the empty dropouts, and counting unique molecular identifiers (UMIs). We used Ensemble hg38/GRCh38 as the reference genome for read alignment. Further analyses including quality control and data filtering, the identification of highly variable genes, dimensionality reduction, standard unsupervised clustering algorithms, and the discovery of differentially expressed genes were performed using the R package Seurat (version 4.1.1) Single Cell 3' Reagent Kits: Chromium™ Single Cell 3' Library & Gel Bead Kit v3, PN-1000075; Single Cell 3' Chip Kit v3 PN-1000073 and i7 Multiplex Kit PN-120262 (10x Genomics). Samples were tagged with cell hashing oligo-tagged antibodies (BioLegend) according to manufacturer's instructions.