{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Qianhui Yu"],"organism":["Homo sapiens"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-17486"],"description":["We infected adult human intestine epithelium stem cell derived colonic chip with wild-type S.Tm 14028 with GFP label. To link intracellular replication levels to host response, we performed single-cell total RNA sequencing (VASA-seq) on index-sorted cells from GFPLow, GFPInt and GFPHigh levels."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Library Construction - For total single-cell total RNA sequencing (VASA-seq), libraries were generated by Single Cell Discoveries (Utrecht, The Netherlands), with the inclusion of a depletion step targeting the S.Tm 5S, 16S, and 23S rRNAs.","Nucleic Acid Extraction - For total single-cell total RNA sequencing (VASA-seq), nucleic acid extraction was performed by Single Cell Discoveries (Utrecht, The Netherlands)","Sample Collection - Cell suspensions were centrifuged at 400 g for 5 min at 4°C prior filtering through 70 µm strainers. For flow cytometric quantification of S.TmGFP-infected cells, fully stained, single stained and unstained samples were resuspended in 250 µl of FACS buffer and analysed using a 5-laser Cytek AuroraTM spectral flow cytometer. Spectral unmixing with autofluorescence extraction was performed using Cytek SpectrofloⓇ CS software, and quantification of GFP+ cell fractions was performed in FlowJo v.10 (BD Biosciences). For FACS, fully stained, single stained and unstained samples were resuspended in 150 µl of FACS buffer. Bulk GFP+ and GFP- populations were isolated using a 5-laser Cytek AuroraTM CS spectral cell sorter equipped with a 100 µm nozzle. Spectral unmixing with autofluorescence extraction was performed using Cytek SpectrofloⓇ CS software. Target cell populations were collected into low protein-binding 1.5 ml microcentrifuge tubes (Eppendorf). Next, the cells were centrifuged, resuspended in FACS buffer, and counted for scRNAseq on the Chromium X.  For total single-cell total RNA sequencing (VASA-seq), single cells were index-sorted using a 5-laser BD FACSAriaTM Fusion with a 100 µm nozzle into individual wells of a 384 well plate filled with lysis buffer (Single Cell Discoveries, Utrecht, The Netherlands). Cells were grouped and sorted by GFP level (GFP-, GFPLow, GFPInt, GFPHigh) to ensure equal representation in the final dataset of each group. Following sorting, plates were immediately stored at -80°C until library preparation. Additional cells were also sorted into a lysis buffer containing 0.5% Triton X-100 (Sigma-Aldrich, T8787) for CFU counting within the same experiment.","Sequencing - For total single-cell total RNA sequencing (VASA-seq), libraries were sequenced by Single Cell Discoveries. Libraries were sequenced aiming for a target coverage of 300,000 raw reads per cell."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - Cells without GFP signal, UMI count < 3500 or ERCC counts < 450 were excluded. Raw counts were normalized using the ‘NormalizeData’ function in Seurat with a scale factor of 10,000.","Sequence Alignment - Reads alignment, gene expression quantification and cell filtering of the VASA-seq data was performed by Single Cell Discoveries (Uthrecht, The Netherlands). In brief, raw sequencing data was processed using Cutadapt (v4.9) to remove adapter sequences and homopolymers, followed by Ribodetector (v0.3.1) to filter out ribosomal reads. Processed reads were then aligned to the reference genome using STARsolo (v2.7.11b), with the parameter --soloFeatures set to GeneFull_Ex50pAS. The human reference genome used was GRCh38, ENSEMBL release 103. The S.Tm reference genome was downloaded from EnsemblBacteria  Salmonella enterica subsp. enterica serovar Typhimurium str. 14028S str. LT2 14028S (GCA_000022165) release 62. The resulting single cell level gene count matrices were used as inputs for downstream analysis in R.  To improve the gene annotation for the S.Tm genome, we performed sequence alignment for the genes in the S.Tm strain 14028S annotation against the S.Tm strain 14028 annotation (GCF_028596185.1_ASM2859618v1). Out of the 1,542 genes with Ensembl-style identifiers beginning with “ENSB”, 242 were mapped to annotated genes with common names in the S.Tm 14028 annotation by ‘blastn’166,167 with more than 90% of sequence similarity."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq X"],"study_type":["RNA-seq of coding RNA from single cells"],"species":["Homo sapiens"],"pubmed_authors":["J.Gray Camp","Qianhui Yu","Stefano Secchia","Rubén López-Sandoval"],"additional_accession":[]},"is_claimable":false,"name":"Single-cell total RNA sequencing (VASA-seq) on index-sorted human colonic chip infected by wild-type S.Tm","description":"We infected adult human intestine epithelium stem cell derived colonic chip with wild-type S.Tm 14028 with GFP label. To link intracellular replication levels to host response, we performed single-cell total RNA sequencing (VASA-seq) on index-sorted cells from GFPLow, GFPInt and GFPHigh levels.","dates":{"release":"2026-08-17T00:00:00Z","modification":"2026-08-17T01:00:43.445Z","creation":"2026-08-13T18:55:37.709Z"},"accession":"E-MTAB-17486","cross_references":{"ENA":["ERP203672"],"EFO":["EFO_0002944","EFO_0004170","EFO_0005684","EFO_0004917","EFO_0005518","EFO_0003816","EFO_0004184"]}}