title: iCLIP-Seq of KIFC1 in prometaphase human Hela cells
design: iCLIP-Seq of KIFC1 in prometaphase human Hela cells
library_construction_protocol: HeLa cells were grown in normal conditions (DMEM high glucose medium, 10% FBS, 37ì§¸C, 5% CO2). 4 biological replicates were subjected to the iCLIP procedure with UV-crosslinking at 254 nm (200 mJ/cm2). We performed iCLIP2 protocol (Buchbender et al, 2020, methods 178:33-48) using anti-KIFC1 antibodies after cell lysis. After immunoprecipitation, the samples were run on a gel and transferred on a membrane. The membrane was cut at 90 to 150 kDa to 4-5 small pieces and transferred into a 2 ml low-bind tube for digestion with proteinase K. Further, RNA extraction was performed using acidic phenol-chloroform-IAA mix and ethanol precipitation. The pellet was resuspended in nuclease free water. For library preparation, we followed the iCLIP2 protocol as detailed in the publication Buchbender et al, 2020, methods 178:33-48 doi: 10.1016/j.ymeth.2019.10.003