GSM8901006: CD45+ cells, Tibialis Anterior, Cardiotoxin dpi4 Control; Mus musculus; RNA-Seq
The isolated GC cells were resuspended in digestion buffer (DMEM with 2%FBS and 10mM of HEPES supplemented with 2 mg/ml collagenase type I and 40 U/ml DNase I) and incubated at 37 °C for 30 min. Cells were washed and re-suspended in MACS Buffer, CD45 enrichment was performed accordingly to the manufacterer reccomendations (Miltenyi Biotec). 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 paired-end or single-end constructs with P5 and P7 sequences and a sample index were added.