GSM4581671: Tmem_He66_S339; Homo sapiens; RNA-Seq
Recombinant HLA-A*02 monomer with UV exchangeable peptide were either synthesized as previously described (Altman and Davis, 2003) or purchased commercially (Biolegend). UV peptide exchange was performed over 20 minutes with 1 mM of peptide in PBS using Strategene UV Stratalinker 2400. Streptavidin conjugated fluorophore was added incrementally the following day for a final 4:1 molar ratio of MHC:streptavidin. Tetramer staining was performed in PBS plus 2% FBS in Fc Blocking solution (Biolegend) at room temperature for 1 hour. For peripheral blood samples, cells were subsequently stained with anti-TCR (B1, Biolegend), anti-CD19 (H1B19, Biolegend), anti-CD14 (M5E2, Biolegend), anti-CD3 (OKT3, Biolegend), anti-CD4 (RPA-T4, Biolegend), anti-CD8 (HIT8a, Biolegend), and live/dead near-IR dye (Invitrogen). Tet+CD8+CD3+CD45+AquaZombie- cells were index sorted directly into 96-well plates preloaded with 4 uL of capture buffer, snap frozen on dry ice, and stored at -80C. Full transcriptomes from FACS sorted T cells at the single-cell level were generated according to the previously reported procedures with some modifications (Picelli et al., 2014). First strand cDNA was then generated with Takara's SMARTScribe Reverse Transcriptase kit according to manufacturer's protocol (Takara Bio). Notable changes from the previously reported Smart-Seq2 RT step includes: 2 mM of dNTP and 2 M of oligo dT were included in the capture buffer; 1M of Betaine and additional 6 mM MgCl2 were included in the RT reaction buffer. The cDNA samples were then amplified with the KAPA Library Quantification kit for 22 – 25 cycles (Roche). We used 1 (of total 25/well) L of amplified cDNA for single-cell TCR-sequencing and thus bypassing the RT step as reported previously (Han et al., 2014). To proceed with scRNA-Seq, full-length cDNA samples were first cleaned up with 0.6 – 0.8x volume of precalibrated AMPure XP beads (Beckman Coulter) to exclude DNA fragments smaller than 500 base pairs. We used the automatic liquid handler Biomek FXP Automated Workstation (Beckman Coulter) in order to eliminate cell-to-cell variabilities. The quality of purified full-length cDNA was validated with the AATI Fragment Analyzer (Agilent). Subsequently, we used the measurements from the Fragment Analyzer in order to normalize the cDNA input with a Mantis liquid handler (Formulatrix). We then consolidated the cDNA samples into a 384-well plate (LVSD) with a Mosquito X1 liquid handler (TTP labtech). After transfer, Illumina sequencing libraries were prepared using a Mosquito HTS liquid handler (TTP labtech). We used only 0.4 uL (of total 23 uL) of cDNA per well to make the full transcriptome libraries with the Nextera XT DNA Library Preparation Kit (Illumina, FC-131-1096). We used custom-made i5 and i7 unique 8-bp indexing primers (IDT) to multiplex 384 wells in a single sequencing run. The libraries were amplified on a C1000 Touch™ Thermal Cycler with 384-Well Reaction Module (Bio-rad). We checked the pooled libraries with the Agilent 2100 Bioanalyzer (Stanford PAN facility) and acquired paired-end sequences (150bp x 2) on a Hiseq 4000 Sequencing System (Illumina) purchased with funds from NIH (S10OD018220) for the Stanford Functional Genomics Facility (SFGF). Smart-Seq2 Method (Picelli et al., 2014)