Illumina HiSeq 4000 paired end sequencing; scRNA-seq of stromal, tumor and immune cells from EMT6 tumors treated with aTGFb/aPD-L1
scRNA-seq of stromal, tumor and immune cells from EMT6 tumors treated with aTGFb/aPD-L1
Tumors were collected 7 days after treatment initiation. Tumors were weighed and enzymatically digested using a cocktail of dispase, collagenase P, and DNaseI for 45 min at 37°C, to obtain a single-cell suspension. Cells were counted using a Vi-CELL XR (Beckman Coulter, Brea, CA). For sorting, cells were first enriched for live cells using Dead Cell Removal Kit following the manufacturer's instructions. Cells were then incubated with mouse BD Fc block (5 µg/ml) for 30 min on ice. And then stained for 30 minutes on ice with the following antibodies: CD45 PE-Cy7 (1 µg/ml), TCR-β APC (1 µg/ml), THY1.1 AF488 (2.5 µg/ml), PDPN PE (0.5 µg/ml), CD31 BUV737 (1 μg/ml), CD11b BUV395 (1 μg/ml),  plus one of the Hashtag antibodies (see attached file scRNA.Barcodes.csv). Just before sorting cells from the same treatment group were pulled together and stained with 7AAD and eBioscience™ Calcein Blue AM Viability Dye. Four population were sorted: T cell (7AAD- Calcein blue+ CD45+ THY1.1- TCRb+), fibroblasts (7AAD- Calcein Blue+ CD45- THY1.1- CD31- PDPN+), myeloid cells (7AAD- Calcein Blue+ CD45+ THY1.1- CD11b+) and tumor cells (7AAD- Calcein Blue+ CD45- THY1.1+). Cells were sorted using a BD FACSAria™ Fusion flow cytometer (BD Biosciences, San Jose, CA). 50k cells for each population were sorted in 300 UL of MACS buffer kept at 4C. Cells were then counted and resuspended at an adequate concentration for loading into the 10X chips. After FACS sorting, cells were processed using the Chromium Single Cell Gene Expression 3′ Library and Gel Bead kit following the manufacturer's instructions (10x Genomics). Cells were counted and checked for viability using a Vi-CELL XR cell counter (Beckman Coulter) and then injected into microfluidic chips to form Gel Beads-in-Emulsion (GEMs) in the 10x Chromium instrument. Reverse transcription was performed on the GEMs, and reverse-transcribed products were purified and amplified. Gene expression libraries and hashtag libraries were made from the cDNA, profiled using a Bioanalyzer High Sensitivity DNA kit (Agilent Technologies), and quantified with a Kapa Library Quantification kit (Kapa Biosystems).