GSM6025874: microglia, ATV:RSV-1-Day (NH24G); Mus musculus; RNA-Seq
Sixteen mice were divided into three batches for processing, each batch contains all five conditions, and a 16th animal was added to the third batch as a control sample.  Mice were perfused with PBS and each hemibrain was dissociated using the Adult Brain Dissociation kit Mouse and Rat (Miltenyi, 130-107-677).  Single cell suspensions were labeled with CD45 MicroBeads Mouse (Miltenyi, 130-052-301) for MACS sorting of microglia.  The enriched microglia cells stored in PBS with 1% BSA were filtered through a 30 μm strainer and counted via hemocytometer for single cell capture using the Chromium Controller (10X Genomics).  Each sample was loaded with a target recovery of 10,000 cells. Single cell libraries were constructed using the Chromium Single Cell 3ʹ Reagent Kits v3 (10X Genomics, PN-1000075).  Each library was barcoded with single indexing using the Chromium i7 Multiplex Kit (10X Genomics, PN-120262), and combined equimolar into a pool for sequencing on a MiSeq sequencer (Illumina) to determine actual single cell capture rate.  A final sequencing pool was combined using the MiSeq cell count values to target a minimum of 20,000 read pairs per cell.  The final sequencing pool was shipped to SeqMatic (Fremont) for sequencing on a NovaSeq 6000 with an S2 flowcell (28X91X8).