GSM6435284: Day 2 Parasites_Astrocyte, scRNA seq; Toxoplasma gondii; RNA-Seq
Toxoplasma tissue cysts were purified from brain homogenate using a percoll density gradient. Tissue cysts were treated with pepsin-HCl mixture to excyst bradyzoites; Parasites that were cultured in human foreskin fibroblast or primary murine astrocytes were needle passed and run through a 3 μm filter on indicated timepoint. All parasites were used for scRNA seq. Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics). Briefly, parasites 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.