GSM5718010: AT_aGC_72hr; Mus musculus; RNA-Seq
Adipose tissue was carefully dissected and dissociated in 1mg/mL Type II collagenase (Worthington) for 25 minutes at 37 degrees celcius. Digested tissue was filtered through a 70uM filter and centrifuged at 15,000rpm for 7 minutes. Spleens were manually disrupted and filtered through a 70uM filter and centrifged. Splenocytes were treated with ACK red blood cell lysis buffer (VWR) prior to further analysis. iNKT cells were FACS-sorted, gated on TCRB+ CD1d-PBS57 Tetramer+ CD19- F4/80- CD45+ live single cells. Single cell suspensions from the adipose tissue and spleens of mice 4 weeks post-αGalCer were tagged by TotalSeq-A Mouse hashtag antibody #2, #3 and #4 (Biolegend) before the three samples were pooled for sequencing. In the HTO sample data hashtag #2 labels adipose iNKT cells, hashtag #3 labels splenic iNKT cells without further stimulation, hashtag #4 labels splenic iNKT cells restimulated with 1μg αGalCer for 4 hours. Cell suspensions were loaded onto a 10x Chromium controller to generate gel bead-in-emulsions (GEMs) according to the manufacturers instructions, and libraries were prepared according to standard protocols produced by 10X Genomics for sequencing using Chromium Single Cell 3' V3 chemistry. Libraries were sequenced using a NextSeq 500 sequencer (Illumina).