title: GSM8768165: sample pool F for TCRseq; Homo sapiens; OTHER
library_construction_protocol: PBMCs were thawed and each PBMC sample was rested for 6h at 37°C with 5% CO2 in 10% medium [RPMI (Gibico, cat. 11875085) + 10% FBS (Gibico, cat. A5670701), and 1% Pen-Strep (Gibico, cat. 15070063)]. Cells were counted and plated in 3 round-bottom wells of a 96-well plate at the density of 1.4 x106 cells in culture with 200 µL 5% medium [RPMI (Gibico, cat. 11875085) + 5% FBS (Gibico, cat. A5670701), and 1% Pen-Strep (Gibico, cat. 15070063)] per well and was stimulated with peanut protein solution (200ng/ul, peanut flour is from Stallergenes Greer cat. XPF171D3A25 and dissolved in PBS) for 18h. Then the cells were washed with Facs buffer (PBS with 0.25% BSA and 1mM EDTA) and stained with Fc block (Biolegend, cat. 422302) following with Single Cell Labeling with the human SMK SmapleTags (BD, cat. 633781) at room temperature for 20 minutes. Cells were surface stained with a panel of fluorochrome-conjugated antibodies including PE-CD154 and PE-CD137 (more details in Supplemental Table 18) in buffer (PBS with 0.25% BSA and 1mM EDTA) for 20 minutes on ice. Then we performed Anti-PE MicroBeads (Miltenyi, cat. 130-105-639) enrichment, following the product instruction to enrich the CD154 and/or CD137 positive cells. After enrichment, we pooled enriched cells from each of the 12 tagged samples to sort PE-positive CD4+ cells, including pr Teff CD4+ T cells (CD4+CD154+) and pr Treg CD4+ T cells (CD4+CD137+CD154-), using a BD FACS Aria at the Stanford FACS facility. We sorted as many pr CD4+ T cells as possible from the enriched cells, yielding between 33,000 and 85,000 cells per set of 12 pooled samples. At the same time, 1000 non-reactive (nr) CD4+ T cells (CD4+CD154-CD137-) were sorted into one single tube from the flowthrough cells from each individual sample. Then, all the pr Teff, Treg CD4+ T cells and nr CD4+ T cells were pooled together following with incubation with AbSeq oligo-linked mAbs pool(Supplemental Table 4, 2ul/mAb in 100ul 2%FBS PBS buffer, all AbSeq oligo-linked mAbs were validated titrated by BD company.). Sorted cells were processed by using the BD RhapsodyTM platform. The libraries were prepared using the BD Immune Response Targeting Panel (BD RhapsodyTM Immune Response Panel HS, cat.633750) with addition of custom gene panel reagents, human TCR primers, BD RhapsodyTM Targeted mRNA and AbSeq Amplification Kit (cat. 633774) and sequenced on Illumina NovaSeq 6000 at Novogene USA (Davis, California).