PacBio capture sequencing of adult human retina
Human donor eyes were obtained from Miracles in Sight (Winston Salem, NC), which were distributed by BioSight (Duke University Shared Resource). Ethical procedures, including procedures for obtaining informed consent from donors, were reviewed and approved by the Duke University Institutional Review Board (protocol #PRO-00050810). Postmortem human donor eyes were enucleated and stored on ice in PBS until dissection. Retinas were dissected from posterior poles and proceeded to RNA isolation. Donors with a history of retinal disease were excluded from the study. Total RNA was isolated using Tri Reagent (ThermoFisher Scientific AM9738) according to the manufacturers protocol. Tissue was mechanically homogenized in Tri Reagent followed by phase separation with chloroform and isopropanol precipitation. RNA samples were stored at -80OC. RIN number was calculated using a Bioanalyzer; only RIN values above 9 were used for sequencing. Reverse transcription was carried out using Clontech SMARTer cDNA kit according to the manufacturers protocol. cDNA was amplified with Prime Star GXL Polymerase for 14 cycles followed by Blue Pippin size selection (4.510 Kb). For capture, 1ug denatured cDNA was used then incubated with Twist Custom Probes at 70C for 20h. Biotynaylated cDNAs were pulled down with streptavidin beads and washed with Twist hybridization buffers to reduce non-specific binding. Targeted cDNA library was amplified 11 cycles with Takara LA Taq yielding 650ng of enriched cDNA for library prep. SMRTbell Template Prep Kit 1.0 post exonuclease was used for library prep followed by a Blue Pippin size selection (4Kb to 50KB). Post size selection yielded 120ng of DNA. Sequencing primer version 3.0 was annealed and bound using polymerase version 2.0. The bound complex was cleaned with PB Ampure beads and loaded onto PacBio Sequel instrument by diffusion at 6pM.