title: RNA-seq analyses of primary human fetal RPE cells (hfRPE) spanning six months in culture
design: RNA-seq analyses of primary human fetal RPE cells (hfRPE) spanning six months in culture
library_construction_protocol: 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.