title: GSM6500159: Brain_mGluR+__Replicate_2; Mus musculus; RNA-Seq
library_construction_protocol: Brain Mononuclear Cell were purified from Toxoplasma (ME49 strain) infected mice. Needle passaged brain tissue was treated with 3mg DNAse and 100μg collagenase/dispase for 1h at 37°C/ 5%CO2. A 30% Percoll density gradient diluted in cRPMI was used to separate cells from myelin layer. Spleens were homogenized through a 40μm strainer using the blunt end of a 5mL syringe.  The homogenate was washed with cRPMI, and 5ml of cold ACK buffer (Lonza) was added for 5min to lyse red blood cells. A cells were prepared for flow cytometry and cell-sorted on CD3+CD8+mGluR1+mGluR5+ and CD3+CD8+mGluR1-mGluR5- T cells. Single Cell RNA sequencing was performed on sorted populations. Library was performed according to the manufacter's instructions (single cell 3' v3.1 protocol, 10x Genomics). Briefly, sorted CD8 T 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.