GSM7065369: HEK 293T cells, TCP seq, DMSO rep 2; Homo sapiens; OTHER
After treatment cells were crosslinked with 0.15% formaldehyde on ice for 10 min with slow agitation. Crosslinking was stopped with 37.5 mM glycine. Cells were washed with PBS and lysed with 100 mM KCl, 20 mM Tris-HCl pH 8, 1.5 mM MgCl2, 1.5% (v/v) NP-40, 2 mM DTT supplemented with protease inhibitor. After lysis, lysate was clarified and digested with Rnase 1 (0.0075 U per µg) for 30 min. Samples was centrifuged on a 4-45% sucrose gradient. RNA corresponding to the 40S fractions was extracted by hot acid phenol extraction. Ribo-seq, Ingolia et al 2012. Reads from 18 to 80 nt were selected.