GSM8912335: CLEAR2_D37_PBMC,RNA; Homo sapiens; OTHER
Previously isolated and viably frozen peripheral blood mononuclear cells (PBMCs) from patient samples were thawed and introduced to prewarmed and filtered 1640 RPMI (Gibco, catalog #11875093) with 10% FBS. Cells were suspended in Human FcR Blocking Reagent (Miltenyi Biotec, catalog #130-059-901) diluted in Filtered DPBS (Corning, catalog #21-031-CM) + 0.5% BSA (Fisher, catalog #F2442-500ML) and incubated for 10 minutes at 4ºC.  Brilliant Staining Buffer (BD), TotalSeqTM Oligonucleotide anti-Human Hashtag antibodies (Biolegend; C0251 catalog # 394661, C0252 catalog # 394663, C0253 catalog # 394665, C0254 catalog # 394667, or C0258 catalog # 394675), surface antibody mix (CD3 PE-Cy7, Invitrogen; CD45 BV510, Biolegend) and the CAR detection antibody (G4S Linker, Cell Signaling Technologies) were added to the FcR Blocking solution and incubated at RT for 30 minutes in the dark. Cells were washed with filtered DPBS + 0.5% BSA, filtered using 30 µm filters (Sysmex), and resuspended in filtered DPBS + 2% BSA + DAPI. Cell populations were purified using Fluorescent Activated Cell Sorting (FACS), using the Aria3 Instrument (BD Biosciences). Upon purification and collection of cell populations in Protein Lo-Bind Eppendorf Tubes, samples were pooled together based off cell number (with a goal of 50-100k cells per sample) and washed with filtered Cell Staining Buffer (Biolegend, catalog # 420201). Cells were Fc Blocked using TruStain FcX (Biolegend) at 4ºC for 10 minutes and then stained with TotalseqTM Universal Cocktail (Biolegned, catalog #399905) for 30 minutes at 4ºC. The cells were washed once with Cell Staining Buffer and twice with DPBS + 0.04% BSA and then submitted to the MSKCC Integrative Genomics Core for sequencing. Approximately 10,000 cells were targeted for each sample (or as many as were available), and several samples were multiplexed together on one lane of 10X Chromium to reach 30,000 cells targeted (when 3 samples were pooled together), following cell hashing protocol. The libraries were prepared using the 10X Single Cell Immune Profiling Solution Kit, according to the manufacturer's instructions. Final libraries were sequenced on Illumina NovaSeq S4 platform (R1 – 26 cycles, i7 – 10 cycles, i5 – 10 cycles, R2 – 90 cycles)