NextSeq 500 sequencing; Combined analysis of single cell RNA-Seq and ATAC-Seq data reveals regulatory toggles operating in native and iPS-derived retina
Combined analysis of single cell RNA-Seq and ATAC-Seq data reveals regulatory toggles operating in native and iPS-derived retina
3D-RA_DD21.2_p
Eyeballs of C57BL/6 wild type mice were enucleated at time points E13, P0, P5 and P9.  Dissected retinas were placed in Dulbecco's Phosphate Buffered Saline (DPBS, Thermo Fisher Scientific). Optic vesicule (OV)-like structures of the iPSCs derived 3D retinal aggregates were cut at DD13, DD21, DD25 and DD29 and transferred in DPBS as well.  Papaïne 4 U/mL (Worthington Biochemical Corporation) was added to the samples. The solution containing the retinas and the OV-like structures was maintained at 37°C for 45 and 30 minutes, respectively.  Ovomucoïde 0,15% (Worthington Biochemical Corporation) was added for papaïne inhibition. Samples were centrifuged in order to eliminate the supernatant and cells were resuspended again in DPBS. Cell numbers and proportion of life cells were estimated by Trypan Blue staining and using a Countess II cell counter (ThermoFisher).  We generated two biological replicates for stages 1 to 3 (NaR and 3D-RA) and one biological replicate for stage 4 (NaR and 3D-RA).   We loaded ~15,700 cells for biological replicate 1 (stage 1-4) and ~10,000 cells for biological replicate 2 (stage 1-3) in a Chromium Controller instrument (10X Genomics, CA).   Sequencing libraries were generated using Chromium Single Cell 3' reagent kits v2.0 following the recommendations of the manufacturer (10X Genomics, CA).