#Robotize file manual

#Robotize file manual

Next, single-cell mRNA sequencing (CEL-Seq or SORT-Seq (robotized version of CEL-Seq2)) was applied, and data was analyzed with RaceID2 to identify clusters.ĬEL-Seq samples: Manual CEL-Seq Satellite cells (unstained) Male Pax7nGFP mice (5-7 months old) 1h collagenase-treated (default dissociation protocol) 96 cells per plate with 96 different barcodes (see 'Cel-seq_barcodes_96.csv') some primes numbers are bulk samples (see 'BulkSamples_BarcodesAndNrOfCellsUsed_perCEL-Seq1-library') Spike-ins included (see 'ERCC92.fa') No mitochondrial reads in count tables Sequencing lanes not concatenated in fastq-files uploaded here 'Merged_CEL-Seq_AllMiceAndLibrariesMerged.csv'-file is count table with reads from all mice and libraries merged (Annotation of columns: Zx.y, where Z = mouse, x = library and y = cell barcode bulk samples are not included any more in this file) See Supplementary Methods for details. For cell extraction, traditional (Supplementary Methods) dissociation protocols that combine mechanical and enzymatic dissociation were employed, and live cells were subsequently sorted into plates using FACS. Mouse satellite cells and zebrafish fin cells were extracted from Tibialis Anterior muscles of Pax7nGFP mice and wildtype zebrafish fins, respectively. Importantly, we detected similar subpopulations in other single-cell datasets, suggesting that cells from other tissues might be affected by this artefact as well. One implication of this finding is that several published transcriptomics studies may need to be reinterpreted. Here, we applied single-cell mRNA sequencing (scRNA-seq) to muscle stem cells, and unexpectedly found a subpopulation that is strongly affected by the widely-used dissociation protocol that we employed. In many gene expression studies, cells are extracted by tissue dissociation and Fluorescence-Activated Cell Sorting (FACS), but the effect of these protocols on cellular transcriptomes is not well characterized and often ignored. Single-cell sequencing reveals dissociation-induced gene expression in tissue subpopulationsĮxpression profiling by high throughput sequencing GEO help: Mouse over screen elements for information.