title: GSM8232691: GRP3__SC_D03_VEH_403_rFP; Mus musculus; OTHER
library_name: GSM8232691
library_construction_protocol: For TRAP immunoprecipitated (IP) samples were prepared by overnight incubation of cell lyate with with protein washed G-coated Dynabeads (Invitrogen) bound to 50 µg of anti-GFP antibodies (HtzGFP-19F7 and HtzGFP-19C8 antibodies). Subsequently, RNA was extracted from the IP and a fraction of the cell lysate (input) samples using Direct-zol RNA kit (Zymo Research). For Ribosome profiling, a fraction of cell clysate was reserved for mRNA-seq while the remaining was used to extract ribosome footprints (rFPs). rFPs were extracted by incubating the cell lysate with 5 µl of RNase I (Ambion, AM2295) per 250 µg of crude total RNA for 45 min at 4 °C and quenched by adding 20 µl of SUPERase-In (Ambion, AM2694) per 5µl of RNase I used followed by ultracentrifugation through a sucrose cushion at 71,000 rpm at 4 °C. Subsequently, RNA was extracted from both the crude cell lysate reserved for mRNA-seq and the resuspended rFP pellet using hot-phenol RNA extraction method with isopropanol based precipitation. rRNA-depleted stranded (HMR)