GSM9701351: N2Ex25, whole bone; Mus musculus; RNA-Seq
To profile the transcriptome of femurs from Notch2em1Ecan and sex-matched control wild type mice, femoral bones from 2 Notch2em1Ecan and from 2 wild type mice were excised after the epiphyseal ends were removed and the marrow was flushed and discarded. Bones were crushed and cells from mice of each genotype were pooled independently and dispersed by digesting the tissue with collagenase, dispase and DNase. Dispersed cells were recovered and incubated with ACK lysis buffer to remove red blood cells followed by fluorescence-activated cell sorting for the depletion of immune and erythroid cells by double-negative (CD45−; TER119−) selection. Sorted cells were suspended and live cells counted and analyzed on a cell-by-cell basis through the use of microfluidic partitioning to capture single cells. Cell partitioning and barcoded libraries were prepared using a Chromium X instrument and a Chromium Single-Cell GEM-X Universal 3' gene expression v4 4-plex library and a GEM-X OCM 3' Chip v4 (10x Genomics). cDNA amplification and library generation were performed according to the manufacturer's protocol and cDNAs from a single-cell had the same barcode, allowing the sequencing reads to be mapped back to the cell of origin. Libraries were validated for length and adapter dimer removal using the Agilent TapeStation D1000 High Sensitivity assay, quantified and sequenced as one sample pool on the AVITI Platform using a 2 x 75 high-output flow cell (AmpSeq). A target read depth of 50,000 transcriptome reads per cell was achieved in each sample.