This README.txt file was generated on <20210527> by ------------------- GENERAL INFORMATION ------------------- 1. Title of Dataset: Supporting data for “Harnessing single-cell transcriptomics analysis to determine cellular heterogeneity, progression, and early markers of conventional chondrosarcoma” 2. Author Information First Author Contact Information Name: SU Zezhuo Faculty: Medicine Email: zezhuo@connect.hku.hk Corresponding Author Contact Information Name: CHEUNG S. C. Kelvin Faculty: Medicine Email: kc81@hku.hk Author Contact Information (if applicable) Name: HO W. K. Joshua Faculty: Medicine Email: jwkho@hku.hk --------------------- DATA & FILE OVERVIEW --------------------- Directory of Files: A. Filename: Support_Data1_filtered_gene_bc_matrices.zip Short description: Processed single cell RNA sequencing data B. Filename: Support_Data2_immunofluorescence.zip Short description: immunofluorescence staining for CHOP and ATF5 in response to ER stress C. Filename: Support_Data3_folw_cytometry.zip Short description: Flow cytometry analysis on cell cyle arrest and apoptosis D. Filename: Support_Data4_wound_healing.zip Short description: SW1353 migration assay in response to ER stress and mupulation of NFKB and Wnt pathways E. Filename: Support_Data5_histology.zip Short description: H&E and Safaranin-O staing on primary tumour tissues F. Filename: Support_Data6_immunohistochemistry.zip Short description: Immunohistochemistry on representative markers of single cell clusters Additional Notes on File Relationships, Context, or Content: file name: "L49", "L07", "L28", "L81", "L63", "L80", "L31", "L44", "L83", "L43" tumour type and grade: "Ben_L49", "Low_L07", "Low_L28", "Low_L81", "Med_L63", "Med_L80", "High_L31", "High_L44", "Ded_L83", "Cos_L43" -------------------------- METHODOLOGICAL INFORMATION -------------------------- 1. 10x library preparation and sequencing Single-cell library and sequencing were performed at the Genomics and Bioinformatics Cores, Centre for PanorOmic Science, Faculty of Medicine, University of Hong Kong. Briefly, single cell encapsulation and cDNA libraries were prepared using Chromium™ Single Cell 3’ Reagent Kits v2/v3 and Chromium™ Single Cell A Chip Kit. Cells were loaded according to standard protocol to capture 5,000 cells to 10,000 cells/chip position per sample. All the remaining procedures including the library construction were performed according to the standard manufacturer’s protocol. Then, library was sequenced by Illumina NovaSeq 6000 using 150 nt paired-end sequencing with 100GB raw reads output per sample. 2. Single-cell RNA sequencing data processing Reads were processed using Cell Ranger 3.0.0 pipeline with default settings. FASTQs generated from Illumina sequencing output were aligned to the human reference genome (GRCh38) using the STAR algorithm (Dobin et al., 2013). Next, we generated gene-barcode matrices for each sample by counting unique molecular identifiers (UMIs) and filtering non-cell associated barcodes. Finally, gene-barcode matrixes containing the barcoded cells and gene expression counts were generated. For the PDX, raw reads were originally aligned to the integrated reference genome of human and mouse (mm10) using standard Cell Ranger pipeline to identify the human cells (83.2%), mouse stromal cells (14.6%), as well as the doublet (2.2%) and characterized the mouse stromal cell types. After identifying the barcodes of human cells, raw reads were aligned to human reference genome only to avoid the impacted of mouse reference genome on the alignment of human gene. Then, the human cells were integrated with cells from primary tumour tissues.