GSM2552193: KO3_RPF; Homo sapiens; OTHER
For ribosome profiling, lysates were subsequently treated with 250 U RNase I (Ambion) for 10 min at 22 °C and shaking at 1400 rpm. The digestion was stopped by addition of 100 U SUPERase In RNase inhibitor (Ambion). Ribosomes were separated on a 10-50 % (w/v) sucrose density gradient in 50 mM Tris pH 7.5, 50 mM NH4Cl, 12 mM MgCl2, 0.5 mM DTT, 100 µg/ml CHX for 3 h at 35 000 rpm and 4 °C in a TH-641 rotor (Thermo Scientific). Gradients were fractionated at 0.75 ml/min with continuous monitoring of OD254 on a density gradient fractionator (Isco) coupled to a SYR-101 syringe pump (Brandel). SDS was added immediately to 1 % and 400 µl fractions were flash-frozen in liquid nitrogen and stored at -80 °C until further processing. Monosome fractions were pooled, RNA isolated and separated on 15 % polyacrylamide gels (8 M urea, 1 x TBE). Ribosome footprints were excised between 28 nucleotide (nt) and 32 nt RNA size markers. Sequencing libraries from ribosome footprints were generated essentially as described (27), except for ligation to a preadenylated 3’-adapter (5’ rAppNNNNCTGTAGGCACCATCAAT/3ddC/ 3’) in a reaction containing 200 000 U T4 RNA ligase 2 (truncated, NEB), 25 % PEG 8000 and 10 U SUPERase In RNase inhibitor for 4 h at 22 °C. The first four bases of the 3’-adapter were randomized to minimize ligation bias (28). Sequencing libraries from ribosome footprints were generated essentially as described (27), except for ligation to a preadenylated 3’-adapter (5’ rAppNNNNCTGTAGGCACCATCAAT/3ddC/ 3’) in a reaction containing 200 000 U T4 RNA ligase 2 (truncated, NEB), 25 % PEG 8000 and 10 U SUPERase In RNase inhibitor for 4 h at 22 °C. The first four bases of the 3’-adapter were randomized to minimize ligation bias (28).
GEO Accession
GSM2552193