GSM8640505: RPE, plastic, P3; Homo sapiens; RNA-Seq
RPE cells were carefully dissociated using a papain solution (20 U/ml) supplemented with DNase-I (2000 U/ml) in Earle's balanced salt solution (EBSS) and incubated in a water bath at 37 °C for 30 minutes. The dissociated single RPE cell pellet was washed with PBS and collected after centrifugation at 1200 RPM for 3 min. The RPE samples were then filtered through a 30 µm cell strainer (Miltenyi Biotech, Cat. #130-098-458) and washed twice with cold Ca²⁺- and Mg²⁺-free 0.04% BSA/PBS at 300g for 5 minutes at 4°C. The samples were gently resuspended in cold Ca²⁺- and Mg²⁺-free 0.04% BSA/PBS and counted using a LUNA-FX7™ Automated Fluorescence Cell Counter (Logos Biosystems) after acridine orange (AO) and propidium iodide (PI) staining (Logos Biosystems, Cat. #F23001). Cells were harvested after 5 days and total RNA was isolated using QIAzol Lysis Reagent (QIAZEN). Total RNA concentration was measured using the Quant-IT RiboGreen Assay (Invitrogen, #R11490). To assess RNA integrity, samples were analyzed using the TapeStation RNA ScreenTape system (Agilent, #5067-5576) and only RNA samples with an RNA Integrity Number (RIN) greater than 7.0 were selected for RNA library preparation. For each sample, 1 µg of total RNA was used to construct libraries using the Illumina TruSeq Stranded mRNA Sample Prep Kit (Illumina, #RS-122-2101). Poly-A mRNA was isolated using poly-T coated magnetic beads and fragmented at high temperatures in the presence of divalent cations. The eyeballs were trimmed to remove surrounding muscle and anterior segments were dissected. After carefully removing the retina, lysis buffer was gently applied to the eye cup and RNA was extracted following the manufacturer's instructions for the RNeasy Mini Kit (QIAGEN). scRNA-seq libraries were prepared using the 10x Chromium Controller and Next GEM Single Cell 5' Reagent v2 kits (10x Genomics, PN-1000244), following the 10x Chromium Single Cell 5' v2 protocol. Cell suspension with a target recovery of 10,000 cells was combined with a reverse transcription master mix and loaded onto a Single Cell K-Chip (10x Genomics, PN-1000286) together with single cell 5′ gel beads and partitioning oil to generate gel beads-in-emulsion (GEMs), in which RNA transcripts from individual cells were uniquely barcoded and reverse transcribed. The resulting barcoded full-length cDNA was amplified by PCR, and for 5' Gene Expression Library preparation, the cDNA underwent enzymatic fragmentation, end repair, A-tailing, adapter ligation, and index PCR. The purified libraries were quantified by the qPCR Qualification Protocol Guide (KAPA), evaluated for quality with the Agilent 4200 TapeStation (Agilent Technologies), and sequenced on the Illumina HiSeq platform (Illumina). cDNA library was generated from the fragmented RNA with SuperScript II reverse transcriptase (Invitrogen, 18064014). Quantification of the prepared libraries was performed using the KAPA Library Quantification Kit for Illumina platforms according to the manufacturer's protocol (KAPA BIOSYSTEMS, #KK4854). Library quality was assessed using the TapeStation D1000 ScreenTape (Agilent, #5067-5582). The indexed libraries were then sequenced on an Illumina NovaSeqX platform (Illumina) with paired-end 2×100 bp reads performed by Macrogen Incorporated. The sequencing library was then constructed using the SMARTer Stranded RNA-Seq Kit according to the manufacturer's instructions. Library quantification, quality assessment, and sequencing on Illumina NovaSeqX platform were performed as described above.