GSM5170228: MIT, Ribotag, 24hrs, Isotype control [E2_M4_30_IP]; Mus musculus; RNA-Seq
60,000 MIT and PMC per sample were lysed in HBS buffer (cyclohexamide 100ug/ml, Protease inhibitor cocktail, Heparin 1mg/ml, RNAsin 1:200, DTT 1mM, NP-40 1%, KCl 100mM, Tris 30mM pH 7.4, MgCl2 82mM). After centrifugation, 10% of supernatant was saved as an input control and 130ul were used for the pulldown of HA-tagged ribosomes using 4ug of anti-HA antibodies or an isotype control (xxxx) (2,5 hrs 4°C). Next, we add 30ul of magnetic Potein A/G Beads (ThermoFisher) for 1 hr at 4°C. Unbound proteins were washed away with high salt buffer (KCl 350mM, NP-40 1%, MgCl2 82mM, DTT 0,5mM, Tris 30mM pH 7.4) three times 10 minutes, 4°C. Precipitated proteins were eluted with 350ul RLT buffer (Qiagen) containing b-mercaptoethanol (1:100).  Subsequently precipitated mRNA species were isolated with a RNeasy Plus Micro Kit (Qiagen) and eluted in 12ul water. cDNA libraries were prepared with 7ul RNA using a Low Input RNA Library Prep Kit for Illumina (NEB).
GEO
GSM5170228