GSM8758452: MYOD1 L122R mutant spindle cell sclerosing rhabdomyosarcoma treated primary resistant; Homo sapiens; RNA-Seq
Tissue dissociation and nuclei isolation from frozen tumor samples were performed using the Singulator 1 device and citric acid preservation. The isolated tumor nuclei were washed in wash buffer containing RNase inhibitor and sorted by FACS using 7AAD as marker. Library was performed according to the manufacturer's instructions (single cell 3' v2 protocol, 10x Genomics). The polyA RNA from the nuclear lysate was retrotranscribed to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10X Barcode. The pooled barcoded DNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriate sized fragments were selected and for subsequent library construction. During library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.