Transcriptional space-time mapping identifies concerted immune and stromal cell patterns and gene programs in wound healing and cancer (Human scRNA-Seq sample dataset))

Published: 26 April 2023| Version 1 | DOI: 10.17632/nnmb6m5p5j.1
Kenneth Hu


Feature, barcode and count matrix for the combined human scRNA-Seq dataset. Samples.csv file denotes patient ID for each cell where HNSC = Head and Neck Squamous Cell and LUNG = Lung Adenocarcinoma. N = Adjacent normal and T = Tumor


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Tumor samples for the Immunoprofiler was transported from cancer operating rooms (ORs). All patients consented by the UCSF IPI clinical coordinator group for tissue collection under a UCSF IRB approved protocol (UCSF IRB# 20-31740). Samples were obtained after surgical excision with biopsies taken by Pathology Assistants to confirm the presence of tumor cells. Patients were selected without regard to prior treatment. Freshly resected samples were placed in ice-cold PBS or Leibovitz’s L-15 medium in a 50 mL conical tube and immediately transported to the laboratory where they were chopped in small pieces (2-3mm2) and place in a cryovial filled with 1mL of freezing media and stored at -80 in a Polycarbonate container, blue high-density polyethylene closure, white high-density polyethylene vial holder overnight. The cryovial were then moved to nitrogen tank for storage. Tumor tissue contained in cryovial were rapidly thawed in a 37C water bath and then tissue were rinsed 3 times in complete media (RPMI 10%FCS) before to be as thoroughly chopped with surgical scissors and transferred to GentleMACs C Tubes (Miltenyi Biotec) containing 20 uL/mL Liberase TL (5 mg/ml, Roche) and 50 U/ml DNAse I (Roche) in RPMI 1640 per 0.3 g tissue. GentleMACs C Tubes were then installed onto the GentleMACs Octo Dissociator (Miltenyi Biotec) and incubated for up to 45min according to the manufacturer’s instructions. Samples were then quenched with 15 mL of sort buffer (PBS/2% FCS/2mM EDTA), filtered through 100 um filters and spun down. Red blood cell lysis was performed with 175 mM ammonium chloride if needed. Cells were then incubated with Human FcX (Biolegend) to prevent non-specific antibody binding. Cells were then washed in DPBS and incubated with Zombie Aqua Fixable Viability Dye (Thermo). Following viability dye, cells were washed with sort buffer and incubated with cell surface antibodies mix diluted (containing anti CD45, anti CD3E, and anti HLA-DR) in the BV stain buffer (BD Biosciences) following manufacturer instruction for 30 minutes on ice in the dark and subsequently washed three time and resuspended in of sort buffer (PBS/2% FCS/2mM EDTA) after filtering in 100um filter (thermo) to be prep for flow sorting. Each sample was then sorted for viable immune cell (CD45+ viability dye -) and viable non-immune cells (CD45- viability dye). Each sample were enriched for immune cells at a ratio of (80% immune 20% non-immune). After sorting, cells were pelleted and resuspended at 1x10e3 cells/ul in 0.04%BSA/PBA and loaded onto the Chromium Controller (10X Genomics). Samples were processed for single-cell encapsulation and cDNA library generation using the Chromium Single Cell 5’ v1.1 Reagent Kits (10X Genomics). The library was subsequently sequenced on an Illumina Novaseq (Illumina). All samples were sequenced at 25,000 reads per cell.


RNA Sequencing