GSM8888243: 2and24moBrain_WTandSTING-KO; Mus musculus; RNA-Seq
Tissues were extracted and placed is appropriate enzymatic digestion buffer for the specific tissue and incubated at 37C. Samples were passed through a 70μm strainer, brought up to 10mL in 2% DMEM, and centrifuged for 5 minutes at 500g at 4°C. Debris was removed from samples using Debris Removal (Miltenyi, 130-109-398) and after cells were pelleted, samples were lysed by resuspension in 1mL ACK lysis buffer (Fisher Scientific, NC0829166) for 1 minute at room temperature, followed by the addition of 9mL of 2% DMEM. Samples were pelleted, resuspended in PBS, and kept cold. Cells were counted (Countessa, Thermo, with Trypan Blue viability stain) and aliquoted for experimental use. Single cell preparations were resuspended into enriched PBS (0.5% BSA). Approximately 9,000 cells were loaded into each well of the Chromium Chip A/B (10x Genomics v2 and v3, respectively), depending on the run, and then run on the Chromium controller. GEM formation quality was visualized manually before GEMs were transferred to a thermal cycler. GEM-RT cleanup and cDNA amplification steps were completed, and quantification was run inhouse. 3' gene expression library as constructed and post-library construction quality control was completed in-house as described in 10x Manual.