Human fetal retinas with no identifiers were obtained from the Birth Defects Research Laboratory under an approved protocol (UW5R24HD000836) through the University of Washington. For whole retina RNAseq and DNA methylation studies, retinas were dissected and stored in Triol (Invitrogen, Carlsbad, CA) at −80C. For immunohistochemistry, whole eyes were fixed in 4% paraformaldehyde for 1 hr at room temperature, cryoprotected in a sucrose gradient, and frozen in OCT (Sakura Finetek, Torrence, CA). Gender was determined by either anatomic identification or PCR. Age was estimated by a combination of clinical intakes, gestational ultrasound, crown-rump, and fetal foot length42 (link),43 (link).
Retinas were dissected at the indicated ages and explanted onto 0.4 µm tissue culture inserts (Millipore, Billerica, MA). For the transcriptome and methylation studies, pairs of retinas were dissected and one was homogenized in Trizol immediately while the other eye was explanted and maintained in Retinal media (DMEM/F12 with GlutaMAX media containing 1% N2, 2% B27, 1% FBS, 1% PenStrep, 1x NEAA, 1x NaPyr, HEPES, and BDNF). For RNAseq studies, retinal explants were collected after 21 days in culture, and for DNA methylation assays, retinal explants were maintained for 42 days. For immunohistochemistry, unpaired retinas were cultured for 21 days.
Retinas were dissected at the indicated ages and explanted onto 0.4 µm tissue culture inserts (Millipore, Billerica, MA). For the transcriptome and methylation studies, pairs of retinas were dissected and one was homogenized in Trizol immediately while the other eye was explanted and maintained in Retinal media (DMEM/F12 with GlutaMAX media containing 1% N2, 2% B27, 1% FBS, 1% PenStrep, 1x NEAA, 1x NaPyr, HEPES, and BDNF). For RNAseq studies, retinal explants were collected after 21 days in culture, and for DNA methylation assays, retinal explants were maintained for 42 days. For immunohistochemistry, unpaired retinas were cultured for 21 days.