GSM8751341: LNI-DDRR-iMN; Mus musculus; RNA-Seq
Intermediate conversion subpopulations at 4 dpi were dissociated by 0.025% trypsin-EDTA. Final converted populations at 14 dpi were dissociated by DNAse (>1,000 U/vial) and papain (>100 U/vial) dissolved into 6 mL DMEM/F-12. Cells were spun down, washed, and resuspended in DMEM/F-12 for FACS isolation. Intermediate conversion subpopulations were identified by low CellTrace-FarRed intensity (hyper-proliferative) at 4 dpi. Hb9::GFP+ at 14 dpi cells were identified relative to Hb9::GFP negative control. 1:1000 Zombie NIR™ was spiked in prior to flow to sort for live cells. Cell subpopultions were then sorted and collected into collection buffer (DMEM/F-12 supplemented with N2, B27, and glutamax) in tubes pre-coated with 7.5% BSA/PBS. Cell suspensions were then spun down and resuspended in PBS. Cell suspension viability was confirmed >90% viable by Trypan Blue staining prior to loading into chip for library construction. Libraries were prepared using 10X Genomics NextGEM 3' v3.1 reagents following manufacturers protocols using 12 cycles of amplification in cDNA generation. Sequencing generated approximately 30-60k reads/cell for each library. Intermediate converting 4 dpi populations had less reads at ~5k reads/cell, presumably due to their lower global transcriptional activity.