Suzanne de Bruijn

81 Homozygous KIAA1549 variants are associated with retinitis pigmentosa KIAA1549 expression in human tissues KIAA1549 expression was determined in human adult tissues using commercially available cDNA panels. Total RNA derived from heart, lung, brain, kidney and bone marrow (Bio-Chain) and total RNA derived from skeletal muscle, liver, duodenum, stomach, spleen, thymus and testis (Stratagene) were utilized. Total RNA from retina was obtained from a healthy anonymous donor. Subsequently, cDNA was prepared using the iScript cDNA Synthesis kit (Bio-RaD) and purified with NucleoSpin Gel and PCR Clean-up Columns (Machery-Nagel). Quantitative PCRwas performed usingGoTaq qPCR Master Mix (Promega) according to manufacturer’s protocol. Transcript-specific intron- spanning primers have been designed and validated for the long (NM_001164665) and short transcript (XM_935390) of KIAA1549 , and for the reference gene GUSB . Primer locations and sequences can be found in Table S1 . Amplifications were performed with the Applied Biosystem Fast 7900 System (Applied Biosystems). All PCR reactions were executed in duplicate and relative gene expression levels compared to the reference gene GUSB were determined with the delta-delta Ct method. Immunofluoresence of KIAA1549 in mouse retinal sections An eye obtained from a healthy 2-month-old mouse was dissected and cryoprotected for 30 minutes with 10% sucrose in PBS before embedding Tissue-Tek OCT (Sakura). Subsequently, sections were frozen in isopentane cooled by liquid nitrogen. For immunofluorescence, unfixed cryosections (7 µm) were permeabilized in 0.01% Tween20 in PBS for 20 minutes. After washing with PBS, blocking was performed for 1 hour using a blocking solution containing 0.1% ovalbumin and 0.5% fish gelatin in PBS. Primary antibodies against KIAA1549 (1:500; cat.# HPA019560, Sigma-Aldrich) and Centrin (1:500; cat.# 04-1624, Millipore) were diluted in blocking solution and incubated on the sections overnight at 4°C. Subsequently, sections were rinsed with PBS and incubated with secondary antibodies goat-anti-rabbit Alexa 568 and goat-anti-mouse Alexa 488 (1:500; Molecular Probes) and DAPI (1:8000; Molecular Probes) in blocking solution for 45 minutes. Finally, sections were post-fixed with 4% paraformaldehyde (PFA) for 10 minutes before mounting with Prolong Gold (Molecular Probes). Sections were analyzed using a Zeiss Axio Imager Z2 fluorescence microscope equipped with an Apotome using several magnifications. Knockdown of KIAA1549 in vitro using siRNAs Silencer® Select siRNAs targeting KIAA1549 (s33562 and s33563) and non-targeting Negative Control No. 1 were obtained from Thermo Fisher Scientific ( Table S2 ). For transfection, hTERT-RPE1 or HEK293T cells (ATCC) were transfected with a single siRNA in duplicate (15 nM final concentration), using Lipofectamine RNAiMax transfection