GSM8572012: MelST Ribo-seq shCntrl 3; Homo sapiens; OTHER
Cells were lysed in ice-cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM MgCl2, 100 mM NaCl, 1 % Triton, 2 mM DTT, 0.25 % Deoxycholate, 100 μg/ml CHX) and cleared for 5 min at 12000 g. For Ribo-seq, 6 A260 units were digested with 36 U RNAse1/unit (10 min, 22°C, 500 rpm) (Ambion, AM2294) and digestion was stopped by adding RNAsin. 10%-50% sucrose gradients were prepared by underlying 50% sucrose dissolved in polysome buffer 2.0 (20 mM Tris-HCl pH 7.5, 10 mM MgCl2, 100 mM NH4Cl, 0.1 mg/ml CHX) a 10% sucrose solution and mixed using a Gradient Master. Digested lysates were loaded onto the sucrose gradients and centrifuged for 3h at 35000 rpm 4°C in a Beckman SW41 rotor. Monosome peaks were collected and RNA extracted. Ribosome protected fragments (RPFs) were PAGE purified on a 15% TBE-Urea geal using PAGExt (Immagina, #KGE002) and library preparation from these fragments was prepared using LACESeq (Immagina, #LS-001) following the instructions of the manufacturer. RNA-Seq libraries were prepared from 1 ug of total RNA. PolyA-selection and library preparation for Illumina.