GSM6605235: GCN1-IP_Untreated(Hsap)_B, Ribo-seq; Homo sapiens; OTHER
C. elegans  Synchronized nematodes were transferred to 1 l S-Basal and grown in liquid culturentil D1 of adulthood or until D6, when indicated. FUdR (200 µM) was added when the culture had reached the L4/young adult stage. After thorough washing in M9, the nematodes were frozen in lysis buffer (25 mM Hepes-KOH pH 7.4, 100 mM KoAC, 10 mM MglCl2, 0.5 mM DTT, 2x EDTA free protease inhibitor, 0.1 U/μL SUPERase*In, 100 μg/mL cycloheximide) by dropwise transfer into liquid nitrogen. The nematodes were lysed with a Retsch Cryomill MM400 at 30 Hz for 3 times 90 s with cooling between cycles. The lysate was cleared for 10 min at 20,000 x g and 4oC. Ribosome protected fragments (RPFs) were recovered, and libraries were prepared as previously described {McGlincy, 2017 #78}.  HEK293T cells  HEK293T cells were seeded into 15 cm plates 24 h prior to treatment. The cells were treated with 10 nM CC-885 and 20 μg/ml G418 for 4 h. Ribosome protected fragments (RPFs) were recovered, and libraries were prepared as previously described {McGlincy, 2017 #78}. C. elegans  The procedure for immunoprecipitation (IP) of GCN-1 bound ribosomes was adapted from {Matsuo, 2021 #75}. Nematodes were synchronized by bleaching. L1 larvae were transferred to 1 L S-Basal. The animals were harvested at D1 of adulthood. Bacteria were removed by repeated washes in M9. In the last washing step, M9 was replaced by lysis buffer (25 mM Hepes-KOH pH 7.4, 100 mM KoAC, 10 mM MglCl2, 0.5 mM DTT, 2x EDTA free protease inhibitor, 0.1 U/μL SUPERase*In, 100 μg/mL cycloheximide). After a quick spin, the worms were flash frozen in liquid nitrogen. The frozen worms were lysed with a Retsch cryomill MM400 at 30 Hz as above. The lysate was thawed on ice and clarified by centrifugation at 4,000 x g for 10 min at 4oC. Protein concentration was determined by Bradford assay. The supernatant fraction (80 mg of total protein) was partially digested with 3 units per 1 mg RNA RNase I (Epicenter) at 4oC for 1 h. Ribosomes were pelleted through a sucrose cushion (1 M, 20 U/mL SUPERase*In (Invitrogen)) at 55.000 rpm in a SW 55 Ti rotor (Beckman Coulter) for 2 h at 4oC and resuspended in lysis buffer supplemented with 10% glycerol. The IP was performed at 4oC for 1 h using M2 anti-FLAG magnetic agarose beads (Invitrogen). The beads were washed 10 times with lysis buffer and bound material was digested with 12.5 units of RNase I (Epicenter) at 23oC for 45 min (strong RNase I treatment). Ribosome protected mRNA fragments were eluted by adding TRIzol reagent (Invitrogen). Libraries were prepared as previously described (McGlincy, 2017 #78) and sequenced on an Illumina NextSeq 500 system.  HEK293T cells  Cells (treated with CC-885 or untreated) were harvested, washed with PBS and lysed by trituration through a 26 G needle for 10 times in lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg/ml cycloheximide, 1 % Triton-X 100). The lysate was cleared at 4,000 x g for 10 min and an equivalent of 5 mg RNA input was digested with 15 units of RNAse I. The digested lysate was then layered over a sucrose cushion (1 M, 20 U/mL SUPERase*In (Invitrogen)) and centrifuged at 55.000 rpm in a SW 55 Ti rotor (Beckman Coulter) for 2 h at 4oC. The pelleted ribosomes were resuspended in lysis buffer supplemented with 10 % glycerol and incubated with GCN1 antibody (A301-843A, Thermo Fisher) coupled to protein G Dynabeads (10003D, Invitrogen) (5 µg antibody per 50 µl of protein G beads) for 2 h at 4oC. The beads were washed 10 times and digested with 4 units RNAse I for 45 min at 23oC. RPFs were isolated by TRIzol and libraries were prepared as previously described (McGlincy, 2017).