title: GSM5782222: GIS2_iCLIP_IME4del_SPO_R3; Saccharomyces cerevisiae; RIP-Seq
library_construction_protocol: For miCLIP, Total RNA was extracted using the hot acid phenol protocol (acid phenol:chloroform:isoamyl alcohol 125:24:1) and TE-SDS buffer, then precipitated in ethanol with 0.3 M sodium acetate before re-suspension in DEPC-treated sterile water. For polyA selection was done with oligodT dynabeads and up to 10 µg polyA RNA was fragmented to around 150-200 n . 4 ug mRNA was incubated with 5 µg anti-m6A for 2h at 4 °C with rotation. polyA RNA was cross-linked twice to the antibody with 2x0.15 J/cm2 UV light (254 nm) in a Stratalinker and then incubated with 30 µl protein G beads for 1.5 h at 4 °C with rotation. For iCLIP and 4-TU-iCLIP, Crosslinked cells were collected and resuspended in lysis buffer and pebbles prepared in liquid nitrogen. Cells were subjected to cryogenic lysis by freezer mill grinding under liquid nitrogen. The resultant yeast “grindate” powder was resuspended in lysis buffer, clarified  and subjected to DNase and RNase treatment before setting up the lysates for immunoprecipitation with V5 antibody.  The immunoprecipitation was carried out overnight at 4 degrees. Immunoprecipitation was followed by 3'end RNA dephosphorylation and adaptor ligation followed by adapter removal. 10% of the sample was labelled with [gamma-P32]ATP  in order to visualize mRNA-antibody complexes by SDS-PAGE and western blotting of both labelled and non-labelled fractions. RNA was extracted from the membrane and subjected to RT reaction and purificatio followed by circularization, PCR and purification of cDNA library before sequencing. For mock miCLIP, the entire protocol was also performed with 1 μg input mRNA omitting IP, P32 labelling, SDS-PAGE/western blotting steps.