GSM7432057: BALB/cJ primary, ENPP1-WT-OE 4T1, biol rep 2; Mus musculus; RNA-Seq
Mammary fat pad primary tumors and lungs containing metastases were harvested from two ENPP1WT-OE or ENPP1T238A-OE tumor bearing mice when primary tumors reached 1000 mm^3. Cryopreserved single cell suspensions were thawed into warm RPMI media with 10% HI-FBS. Cells were washed with PBS and 0.04% w/v BSA, passed through 40 m Flowmi cell strainer (Bel-Art, 974-25244), and assessed for concentration and viability with an automated cell counter. Viability varied between 72-87% for tumor samples and 91-96% for lung samples. Cells were resuspended in PBS and 0.04% w/v BSA to 1000 cells/uL. Library was performed according to the manufacter's instructions (single cell 3' HT reagent kits v3.1 protocol, 10x Genomics). Briefly, cells 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.