GSM8060616: As4.1 cells treated with 10 μM H89; Mus musculus; RNA-Seq
600,000 cultured As4.1 cells were plated in duplicate in T25 flasks.  The cells were treated with 10 μM H89 or equivalent volume DMSO at 0, 24, and 48 hours. After 48 hours, cells were trypsinized and 4000 cells per condition were targeted for sequencing. The cells were centrifuged in a Sorvall RT7 refrigerated 4∘C centrifuge (Sorvall, Newtown, CT) at 500 x g for 10 minutes, washed 3X with 5mL of dPBS, then 1 mL of trypsin was added, and cells incubated at 37∘C for 2 minutes before adding 4 mL of fresh media.  The cells were gently pipetted up and down 20 times to dissociate and centrifuged at 800 x g for 3 minutes at room temperature.  The supernatant was removed, and pellets resuspended in 1.5mL dPBS supplemented with 0.04% BSA (UltraPureTM Bovine Serum Albumin, Invitrogen, Lithuania) by pipetting up and down 20 times.  Resuspended cells were passed through a 40 μm filter and repelleted by centrifugation for 3 minutes at room temperature at 150 x g.  The supernatant was carefully removed, and cells washed 3X in 1.5mL dPBS with 0.04% BSA as outlined above.  Cells were resuspended a final time in 1.5mL dPBS with 0.04% BSA and the volume adjusted to yield 1000 cells/μL. Isolated single cells were loaded and captured using the Chromium System (10X Genomics, Pleasanton, CA) following the manufacturer's recommendation for the Chromium Next GEM Chip G with reagents of Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (10X Genomics).