title: Illumina HiSeq 3000 sequencing; ChIP-seq to investigate the phase separation-dependent FUS RNA interactome
design: ChIP-seq to investigate the phase separation-dependent FUS RNA interactome
library_construction_protocol: HEK293T cells expressing wild type or P525L eGFP-tagged FUS fusion protein were lysed and subsequently subjected either to a co-IP experiment using anti-GFP nanobodies coupled to magnetic beads or to eGFP-FUS droplet purification. For co-IP, the lysates were subjected to GFP nanobodies coupled to magnetic beads after immunoprecipitation and subsequent washes, 1 ml TRIZOL was added directly to the beads for RNA isolation. Droplets were generated through reducing the volume of the lysate and stabilized using the reversible crosslinker formaldehyde. Thereafter, the droplets were purified by fluorescence activated particle sorting and additional wash steps and eluted in RNA-sample buffer (50 mM TRIS pH7.0, 5 mM EDTA, 1 % SDS, 10 mM DTT). To isolate RNA from the droplets, the formaldehyde crosslink was reversed through incubation of the droplets in RNA-sample buffer (50 mM TRIS pH7.0, 5 mM EDTA, 1 % SDS, 10 mM DTT) for 40 min at 70 °C. After cooling of the sample on ice for 5 min, 1 ml TRIZOL was added.  HEK293T cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10 % fetal calf serum (FCS), penicillin (100 U/µl) and streptomycin (100 µg/ml)) at 37 °C and 5 % CO2. RNA was isolated from TRIZOL according to the manufacturer's instructions Quality and quality of RNA was analyzed with an Agilent 2100 Bioanalyzer (Agilent Technologies). Total RNA isolated from transiently transfected cells served as input RNA. Total RNA isolated from droplets, from the co-IP and input RNA (in biological triplicates for each sample) were ribodepleted using the RiboMinus™ Transcriptome Isolation Kit (Invitrogen, K155004) before library preparation according to the manufacturer's manual. Libraries were prepared using the strand-specific Illumina TruSeq Stranded Total RNA kit (Part # 15031048 Rev. E).