title: GSM5600150: Bulk-RNA-seq_KO-RPE-P30-1; Mus musculus; RNA-Seq
library_construction_protocol: Sc RNAseq:  Eyes were enucleated for retina tissue isolation. After removing the anterior chamber and lens, retinal cells were dissociated using the Papain Dissociation System (#LK003153, Worthington Biochemical) following the manufacturer's instructions. In each of the six experimental groups (WT and AdipoR1-/- at P19, P25, and P30), four retinae were used to prepare single-cell suspensions at a final concentration of 1,000 cells/µl.    Bulk RNAseq:   Eyes from AdipoR1-/- and WT mice at P30 were enucleated and placed in 60 mm dishes with PBS immediately following cervical dislocation. The anterior segment of the eye, including cornea, iris, and lens, was removed using dissecting scissors. The retinas from both eyes were carefully removed and transferred to 400 µl of RNAlater stabilization solution (#AM7920, Thermo Fisher). The posterior eyecups, consisting of RPE, choroid, and sclera, were transferred to 400 µl of RNAprotect tissue reagent (#76104, Qiagen). Eyecups were incubated at room temperature for 10 min and briefly agitated to detach RPE cells from the choroid/sclera. After removing the choroid-sclera, the remaining RPE cells were pelleted by centrifugation for 5 min at 685 x g. The isolated retinas and RPE cells in stabilization solution were subjected to extraction of total RNA using RNeasy Micro Kit (#74004, Qiagen) following the manufacturer's protocol. Sc RNA-Seq:   For each group, freshly dissociated cells (~10,000) were loaded into a 10x Genomics Chromium Single Cell system using v2 chemistry following the manufacturer's instructions. In the first batch (B1: WT and AdipoR1-/- at P30), two libraries were pooled and sequenced on Illumina NextSeq500 with ~76 million reads per library. In the second batch (B2: WT and AdipoR1-/- at P19 and P25), four libraries were pooled and sequenced on Illumina NovaSeq6000 with ~143 million reads per library.  Bulk RNAseq:   Total RNA (~100 ng per sample) was used for library preparation by following the Illumina TruSeq Stranded Total RNA Library Prep Kit protocol. Libraries were then sequenced (paired-end 100 cycles) with the Illumina NextSeq 500 platform, yielding approximately 40 million raw reads per library.