GSM7927081: SW480, shEIF2B1, induced, repl 1 [2B1_3A_plus_RPF_1]; Homo sapiens; RNA-Seq
Pellets were resuspended in non-denaturing lysis buffer (15 mM Tris-Cl pH 7.5, 150 mM NaCl, 15 mM MgCl2, 100 μg/ml cycloheximide, 1% Triton X-100, 0.05% Tween-20, 2% n-Dodecyl-beta-Maltoside Detergent (DDM), 0.5 mM DTT, 5 mM NaF, 1x cOmplete mini (#04693124001, Roche)). For RPFs: Lysates were digested with 8 μl of RNAseI (#AM2295, Lot# 00791236, Ambion) at 22°C for 20 min. Digestion was stopped with 16 μl of SUPERaseIn (#AM2696, Invitrogen). Digested lysates were run on a 10-50% sucrose gradients where fractions corresponding to monosomes and disomes were collected. RNA from these fractions was extracted by acid phenol-chloroform extractions, further processed to select for ribosome protected frament (RPF) size, treated with T4 Polynucleotide Kinase (NEB M0201S) and size checked on an Agilent Bioanalyzer small RNA chip. For total cytoplasmic RNA: prior to digesting the lysate for RPF extraction, an aliquot was kept aside and mixed with 1 ml of TRIzol™ Reagent (Invitrogen, 15596026). RNA extraction from TRIzol was performed according to manufacturer's instruction, with a further Ethanol precipitation to eliminate all impurities. Average eRIN value of total cytoplasmic RNAs was 9.7, as checked by RNA screen tape on a TapeStation (Agilent) Total cytoplasmic RNAs were depleted of rRNA with RiboCop v2 (Lexogen) and then libraries were prepared using the CORALL kit (Lexogen), all following the manufacturer's instructions. 900 μg of total cytoplasmic RNAs were used as input for the RiboCop. 13 PCR cycles were used in the library amplification stage. For RPFs: Libraries were prepared with the NEXTflex small RNA kit v3 (Perkin Elmer) following the manufacturer's instructions, using 10.5 ng of input and 11 PCR cycles in the amplification step. Alternative step F and Step H2 were used.