scRNA-seq of homo sapiens: induced pluripotent stem cells
Samples were prepared as described by the 10x Genomics Cell Multiplexing Oligo Labeling for Single Cell RNA Sequencing Protocols for cells with >80% viability. Cells were labeled with CellPlex Multiplexing Solution from 10x genomics with a sample-unique oligo barcode for multiplexing with other samples. Following a final counting and viability analysis, cells and 10x Genomics reagents were loaded into the single cell cassette, with a target of 25000 single cells for analysis, accounting for predicted cell loss and doublets resulting from multiplexing as laid out in the user guide for the Chromium Single-Cell 3 Reagent Kit (10x Genomics). After generation of GEMs, the cDNA library was prepared by Pitt Single Cell Core staff following the appropriate steps determined by the 10x Genomics user guide. Libraries were sent to the UPMC Genome Center for sequencing by a NovaSeq S4-200 for an intended read depth of 100,000 reads per cell with 150 bp paired end reads. Our downstream analysis from the sequencing data yielded between 40k-150k mean reads per cell in different samples. The 10x Genomics CellRanger pipeline was used to align reads to the reference genome (GRCh38.84) appended with transgene sequences, to assign reads to individual cells, and to estimate gene expression based on UMI counts. BAM files were created from the multiplexed fastq files, and these BAM files were converted back to Fastq files via the "bamtofastq" function in CellRanger.