RNA-seq analyses of primary human fetal RPE cells (hfRPE) spanning six months in culture
RNA-seq analyses of primary human fetal RPE cells (hfRPE) spanning six months in culture
After removing the culture media, lysis buffer was added on top of the cultures, then lysed cells were transferred to RNase-free eppendorf tubes.  The RNA of a total of 11 samples (3 biological replicates of each age, except for 25 weeks, with 2 biological replicates) was extracted using RNeasy Mini Kit (Qiagen), according to the manufacturerâ€™s protocol mRNA enrichment and purification was performed using oligo(dT) coupled to magnetic beads. cDNA library construction involved mRNA fragmentation, cDNA synthesis (generating first-strand cDNA with random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis), end-repair, A-tailing and adapter ligation, followed by PCR amplification, circularization and rolling circle amplification for DNA nanoball (DNB) generation, which harbored more than 300 copies of one molecule.