GSM8003561: Tumor implants from TrkA^WT;NOD-Scid, pooled triplicates, sc-RNAseq; Mus musculus; RNA-Seq
After collagenase digestion, enzyme activity was diluted by adding PBS. Cell fractions were collected and resuspended in red blood cell lysis buffer (37°C for 5 min). Digestions were sequentially filtered through 100-μm and 40-μm sterile strainers. Cells were then washed in PBS and resuspended in HBSS at a concentration of ~1000 cells/μl. Cell viability was assessed with Trypan blue exclusion on a Countess II (Thermo Fisher Scientific) automated counter and showed a >85% viability. Cells were sent to JHMI Transcriptomics and Deep Sequencing Core. The library was generated using the 10x Genomics Chromium controller following the manufacturer's protocol. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.