title: GSM8794789: VEEV_7_DPI; Mus musculus; RNA-Seq
library_construction_protocol: From the right hemisphere, the hippocampus was dissected and placed into 10x genomics fixation buffer solution (Chromium Next GEM Single Cell Fixed RNA Sample Preparation Kit #1000414, 10x Genomics, Pleasanton, CA). N=5 pooled hippocampus was minced in a petri dish until they could be pipetted through a wide bore pipette tip and weighed to achieve 30mg total fixed tissue. After fixation, the pieces were spun down and washed with PBS following 10X sample fixation protocol and stored at 4°C and shipped to Medgenome, Foster City, CA. At Medgenome, Fixed and quenched tissues were dissociated for 10 minutes at 37°C using an Miltenyi Octo Dissociator (Mitenyi Biotec, Gladbach, Germany #130-096), dissociated tissue were filtered through a 30 um filter to remove debris and undissociated tissue pieces, and centrifuged at 850 rcf for 5 minutes. The cell pellet was resuspended in chilled quenching buffer. Cells were stained with ethidium homodimer-1 and counted using countess II FL. The cell concentration was determined based on the reactive fluorescent protein positive concentration. For custom probe spike in, single cell gene expression flex probe configuration with sequences (Table 4) ordered from Integrated DNA Technologies (Integrated DNA Technologies, Coralville, IA). Each probe represents 25 bp sequences homologous to the target transcript (VEEV genome identified from NCBI GenBank: L01443.1). One – two million cells were hybridized with a modified probe hybridization mix containing mouse WTA probes BC001 and custom probes, each probe at 40 nM for 16-24 h at 42°C. Each Left-hand probe (LHS) contained a linker region 5'-CCTTGGCACCCGAGAATTCCA-target_LHS-3' and right hand probe /5Phos/-target_RHS-ACGCGGTTAGCACGTANNACTTTAGGCGGTCCTAGCAA-3'. Following probe hybridization, gel beads in emulsions were generated for 6000 cells targeted recovery using 10x genomics chromium fixed RNA profiling reagent kit (10X Genomics, Pleasanton, CA, #1000414). The libraries were generated and sequenced in NovaSeq X for 120M paired reads.