NextSeq 500 paired end sequencing; ATAC-Seq using native and iPSC-derived murine retina at three developmental stages.
ATAC-Seq using native and iPSC-derived murine retina at three developmental stages.
ATAC_NaR_P0.1_1ul_p
Eyeballs of C57BL/6 wild type mice were enucleated at time points E13, P0, and P5.  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, and DD25 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 using a Countess II cell counter (ThermoFisher).  We generated two biological replicates. We used 50,000 cells per tagmentation reaction. NA ATAC-seq libraries were constructed by following the Omni ATAC protocol (Corces et al., 2017). We tested two different amounts of the Tagment DNA TDE1 enzyme (1 and 2 μl in a 50 μl reaction volume) (Illumina, San Diego, California) per sample. Genomic DNA (gDNA) libraries were also prepared using 50 ng of gDNA isolated from NaR P5 and 3D-RA DD25 cells by following the Nextera DNA Sample Preparation Guide (Illumina). The libraries were purified using the MinElute PCR purification kit (Qiagen, Venlo, Netherlands) followed by 13 and 5 cycles of PCR-amplifications for ATAC-seq and gDNA libraries, respectively. After validating library size distribution using the QIAxcel capillary electrophoresis (Qiagen), the libraries were further purified using the SPRIselect reagent to remove large DNA molecules (a right-side size selection with 0.55X followed by 1.5X ratios of beads) (Beckman Coulter, Brea, California).