Dynabeads myone streptavidin t1 beads

In this study, 269 cCSC patients who visited the outpatient clinic of the Department of Ophthalmology of the Radboud university medical center, Nijmegen, the Netherlands were included. Patients were diagnosed with cCSC based on multimodal retinal imaging as described previously^{6 (link),10} . DNA of cCSC patients, isolated from blood, was purified with the QIAamp DNA purification kit (Qiagen), according to manufacturer’s instructions. 3 µg of purified DNA was used as input for the SureSelect^XT target enrichment system for Illumina paired-end multiplexed sequencing (Version B4, August 2015, Agilent Technologies).
Library preparation was performed according to the manufacturer’s instructions. Samples were hybridized with the SureSelect All Exon V4 (Agilent Technologies) capture library and post-hybridization addition of the index tags was performed on the Dynabeads MyOne Streptavidin T1 beads (ThermoFisher Scientific). Midway and final quality checks were performed with Tapestation high sensitivity D1000 screentape (Agilent Technologies) and a Qubit dsDNA high sensitivity assay (ThermoFisher Scientific). A total of 8 samples was pooled for sequencing in one lane. Sequencing was performed at the Department of Genetics of Maastricht University Medical Center+, Maastricht, The Netherlands, using an Illumina HiSeq2000 with 2*100 bp chemistry.

We used the in situ Hi-C protocol as described in Rao et al.^{48 (link)}. In brief, ~3 × 10⁶ RPE cells were crosslinked with 1% formaldehyde for 10 min at room temperature, followed by an additional 5 min with 200 mM glycine in phosphate-buffered saline (PBS). Fixed cells were permeabilized in Hi-C lysis buffer (10 mM Tris–HCl, pH 8.0; 10 mM NaCl; 0.2% Igepal CA630; 1× protease inhibitor cocktail [Sigma]) on ice. The cells were treated with 100 U of MboI (New England Biolabs) for chromatin digestion, and the ends of digested fragments were labeled with biotinylated nucleotides followed by ligation. After DNA reverse crosslinking and purification, ligated DNA was sheared to a size of 300–500 bp using a Covaris S2 focused-ultrasonicator (settings: Duty Cycle, 10%; Intensity, 4; Cycles per Burst, 200; Duration, 55 s). The ligated junctions were then pulled down with Dynabeads MyOne Streptavidin T1 beads (Thermo Fisher Scientific). The pulled-down DNA was end-repaired, ligated to sequencing adaptors, amplified on beads, and purified using a Nextera Mate Pair Sample Preparation kit (Illumina) and Agencourt AMPure XP (Beckman Coulter). DNA was then sequenced to generate paired-end 150-bp reads using the Illumina HiSeq-2500 or X Ten system.

Hi-C libraries were processed with our in-house pipeline based on the previously published in situ protocol (Rao et al. 2014 (link)). Briefly, ~ 1 million cells were fixed in 2% formaldehyde, lysed and digested overnight with DpnII enzyme (New England BioLabs). Next, digested DNA ends were marked with biotin-14-dATP (Thermo Fisher Scientific) and ligated overnight using T4 DNA ligase (New England BioLabs). DNA was sheared to fragments of 300–500 bp for library preparation and biotin-filled DNA fragments were pulled down using Dynabeads MyOne Streptavidin T1 beads (Thermo Fisher Scientific). The DNA was prepared for short reads sequencing by ligating adaptors to the DNA fragments, using the NEBNext Multiplex Oligos for Illumina kit (New England BioLabs). Libraries were deep sequenced (~ 240 Million fragments) in a 75 bp paired-end run on a HiSeq4000 (Illumina). Paired-end sequencing data were processed using the Juicer pipeline (Durand et al. 2016 (link)). A detailed protocol is described elsewhere (Melo et al. 2020 (link)).