2.4 RNA isolation and PacBio library preparation

To obtain the total RNA from human retina samples, 500 µL of Trizol was added and each sample was homogenized in two rounds using a Tissuelyser kit (QIAGEN, Aarhus, Denmark) for 30 s at 30 Hz. After a 5-minute incubation at room temperature (RT), 100 µL chloroform was added, samples were mixed, incubated for 3 min at RT, and centrifuged at 12,000 g for 15 min. Afterwards, the aqueous phase was mixed with glycogen (5 μg/μL) and 1 volume of isopropanol, and the resulting mixture was incubated at 20°C for 75 min and subsequently centrifuged at 12,000 g for 30 min at 4°C. The supernatant was discarded, and the resulting RNA pellet was further purified and DNAse treated using the Nucleospin RNA Clean-up Kit (Macherey-Nagel, Duren, Germany) according to the manufacturer’s protocol. The total isolated RNA was quantified using a Qubit fluorometer (Thermo Fisher Scientific, Waltham, MA, United States) and RNA integrity number (RIN) values were assessed using a 2,100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, United States). The RIN values of the three samples were above 7.0 and 300 ng of RNA input was used to generate the Iso-Seq SMRTbell libraries using the Iso-Seq-Express-Template-Preparation protocol version 2.0 (Pacific Biosciences, California, United States). Libraries were prepared following the standard workflow (for samples composed primarily of transcripts centered ∼2 kb) and using the SMRTbell^® library binding kit 2.1 (Pacific Biosciences, California, United States). The on-plate loading concentration of the final Iso-Seq SMRTbell libraries was 80 pM, and a 24-h movie time was used for sequencing on a Sequel II system (Pacific Biosciences, California, United States).