Spatial centrosome proteome of human neural cells uncovers novel interactors and disease hubs

Version 1

RNA from FACS sorted electroporated (GFP+) cells was isolated in Extraction Buffer (Arcturus), heated to 42 and stored at -80 cDNA was synthesized from 300 pg of total RNA using SMART‐Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech), according to the manufacturer's instructions. Prior to generating the final library for Illumina sequencing, the Covaris AFA system was used to perform the cDNA shearing, resulting in 200‐ to 500‐bp‐long cDNA fragments. The quality and concentration of the sheared cDNA were assessed on Agilent 2100 Bioanalyzer before proceeding to library preparation using MicroPlex Library Preparation kit v2 (Diagenode). Final libraries were evaluated and quantified using an Agilent 2100 Bioanalyzer, and the concentration was measured additionally with Quant‐iT PicoGreen dsDNA Assay Kit (InvitrogenTM) before sequencing. The uniquely barcoded libraries were multiplexed onto one lane and 150‐bp paired‐end deep sequencing was carried out on HiSeq 4000 (Illumina) that generated ~ 30 million reads per sample.