GSM5509915: Naïve ear  [high.W6.Naive]; Mus musculus; RNA-Seq
At 4 week, with or without infection, mice ears were excised and enzymatically digested using 0.25 mg/ml Liberase and 10 mg/ml Dnase-I in incomplete RPMI 1640 for 90 minutes at 370 C. After digesting, ears were minced manually to obtain cellular content in single cell suspensions. The total RNA was isolated from the cell lysate of naive and infected ears by using RNeasy plus mini kit according to the manufacturer's instruction. Single cell 3' library generation was performed using a 10X Genomics Chromium Controller and the v3 reagent kit (10x Genomics) according to the manufacturer's protocol.  Cell suspensions were loaded on to the middle four channels of a Chromium Single Cell A Chip according to the manufacturer's instructions, aiming for 10,000 cells per channel.  Following generation of single-cell gel bead-in-emulsions (GEM), reverse transcription, fragmentation and cDNA amplification, library preparations and bar coding were performed using the Illumina TruSeq Stranded mRNA Sample Preparation Kit v2 as per manufacturer's instructions 200pM of each library was pooled for sequencing on the Illumina NextSeq 500 platform using a high output flow cell (100 cycles) to generate approximately 25 million 75 base reads per sample.