GSM7057457: Infected, replicate 1, scRNAseq; Mus musculus; RNA-Seq
Mice were infected ocularly with HSV-1 McKrae and TG were harvested on day 35 PI. Thirty TG from 15 latently infected mice were pooled and washed with cold 1x HBSS six times, then incubated with 2 mg/ml collagenase D at 37oC degree for 1 hr to generate single cells. Dead cells were removed using a dead cell removal kit from STEMCELL (Seattle, WA, USA) according to the company protocol. CD45 positive cells were isolated using a mouse CD45 positive cell selection kit from STEMCELL according to company protocols. Isolated CD45 positive cells were mixed back with the original single cell suspension at a 1:1 ratio and used for single cell sequencing library preparation. Single-cell RNAseq libraries were prepared according to the Single Cell 3′ v3.1 Reagent Kits User Guide (10x Genomics, Pleasanton, California). Cell suspensions were loaded on a Chromium Controller instrument (10x Genomics) to generate single-cell Gel Bead-In-EMulsions (GEMs). GEM-RT was performed in a Veriti 96-well thermal cycler (Thermo Fisher Scientific, Waltham, MA). GEMs were then harvested, and the cDNA was amplified and cleaned up using a SPRIselect Reagent Kit (Beckman Coulter, Brea, CA). Indexed sequencing libraries were constructed using a Chromium Single-Cell 3′ Library Kit for enzymatic fragmentation, end-repair, A-tailing, adapter ligation, ligation cleanup, sample index PCR, and PCR cleanup. The barcoded sequencing libraries were quantified by quantitative PCR using a Collibri Library Quantification Kit (Thermo Fisher Scientific).