title: GSM7874577: APRIL-BB-1XX_CITE; Mus musculus; OTHER
library_name: GSM7874577
library_construction_protocol: CAR T-cell treated mice were harvested for bone marrow on day 7 of experiment (4 days after CAR T-cells). Cells were erythrocyte-lysed and stained with Fc-block. Cells were then labeled with fluorescent antibodies and DAPI for FACS sorting and hashtag oligonucleotide (HTO) antibodies (Biolegend) to barcode each mouse for identification post-sequencing. Cells were FACS-sorted for CD45+/CD3+ T-cells and CD45+/CD3- non-T-cells. Cells were re-pooled at 1:2 T- cells:non-T-cells and stained with FcX (Biolegend) followed by Biolegend TotalSeqA mouse universal cocktail (for CITE-seq). ~100,000 cells from each biological group were submitted for single-cell sequencing (n=3 mice per group, ~30,000 cells /mouse). Single-cell RNA sequencing of FACS-sorted cell suspensions was performed on the Chromium instrument (10X genomics) following the user guide manual for 3′ v3.1. In brief, FACS- sorted cells were washed once with PBS containing 1% bovine serum albumin (BSA) and resuspended in PBS containing 1% BSA to a final concentration of 1,700–2,000 cells per μl. The viability of cells was above 80%, as confirmed with 0.2% (w/v) Trypan Blue staining (Countess II). Cells were captured indroplets. Following reverse transcription and cell barcoding in droplets, emulsions were broken, and cDNA was purified using Dynabeads MyOne SILANE followed by PCR amplification as per manual instruction. About 30,000 cells were targeted for each sample. Samples were multiplexed together on one lane of 10x Chromium (using HTO) and specific extracellular protein epitope characterized (using Antibody Derived Tag, Biolegend TotalSeqA) following a previously published protocol3. Final libraries were sequenced on Illumina NovaSeq S4 platform (R1 – 28 cycles, i7 – 8 cycles, R2 – 90 cycles) at a depth of ~575 million reads per sample. HTO and CITE were sequenced at ~88 million reads per sample.