biostudies-arrayexpressKanishk AsthanaHomo sapiensChronoSeq-Toolshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15947We applied ChronoSeq to profile inflammatory responses in K562 cells stimulated with TNF-α, to capture the rapid NF-κB signaling cascade. 10 minute interval between samples including a 1 minute sampling duration. In addition Puromycin was added at a does of 5μg/ml for blocking translation. The purpose of this experiment was to measure TNF-α stimulation transcriptional program without translational feedback.biostudies-arrayexpressNucleic Acid Extraction - Oligo-DT beads were used to capture mRNA from lysed Single-Cells inside droplets.Growth Protocol - K562 cells grown in suspension in DMEM with 10% FBS and 1%PEN-STREP at 5%CO2 and 37C. https://kanishkasthana.github.io/ChronoSeq/protocol_for_preparing_cells_AdditionalFiltration.htmlSample Collection - Automated cell sampling using ChronoSeq device. Each cell sample is injected with a unique injection of Time-Tagged ChronoSeq beads. https://kanishkasthana.github.io/ChronoSeq/protocol_and_software_for_running_chronoseq_device.htmlSequencing - Illumina NovaSeq X 10B on one lane in PE100 configuration with Custom Read 1 primerSample Treatment - 10ng/ml TNF-α + 5μg/ml Puromycin were added to stimulate the cells and block translation respectively. Both were added together 5 minutes after first sample.Library Construction - ChronoSeq library preparation protocol is very similar to Drop-seq. Beads from all time-tags are pooled together, then split into four equal parts for Reverse Transcription followed by PCR amplification. https://kanishkasthana.github.io/ChronoSeq/protocol_library_preparation_for_dropseq_chronoseq_beads.htmlOrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - hg38 alignment. Data processed using ChronoSeq-Tools pipeline. https://github.com/kanishkasthana/ChronoSeq-ToolsData Transformation - UMIs and gene information were used to generate unique transcripts captured and remove PCR duplicates using Drop-seq tools.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq XRNA-seq of coding RNA from single cellsHomo sapiensKanishk AsthanafalseK562 stimulated with TNFα with translation blocking using Puromycin. 12 samples were taken with ChronoSeq and barcodes with unique time-tagsWe applied ChronoSeq to profile inflammatory responses in K562 cells stimulated with TNF-α, to capture the rapid NF-κB signaling cascade. 10 minute interval between samples including a 1 minute sampling duration. In addition Puromycin was added at a does of 5μg/ml for blocking translation. The purpose of this experiment was to measure TNF-α stimulation transcriptional program without translational feedback.2025-11-01T00:00:00Z2026-05-26T12:05:31.759Z2025-10-31T21:14:10.464ZE-MTAB-15947ERP183478E-MTAB-15927E-MTAB-15928E-MTAB-15894EFO_0002944EFO_0004170EFO_0003789EFO_0005684EFO_0004917EFO_0005518EFO_0003816EFO_0004184EFO_0003969