title: GSM2935911: mES KO postHS_1; Mus musculus; OTHER
library_construction_protocol: Ribosome protected fragment  were obtained aw previously described by McGlincy, N.J. and Ingolia N.T. Methods 126, 112-129 (2017).  Briefly, to inhibit ribosome transit post-lysis, cells were rapidly washed twice with ice-cold PBS containing 50 µg/ml of cycloheximide. To generate ribosome-protected fragments, cells were pelleted and immediately lysed in 400 µL of cell lysis buffer (20 mM Tris pH 7.4, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 100 µg/mL cycloheximide, 25 U DNase I). 30 µg of RNA were used to isolate ribosome-protected fragments following RNase I digestion. After RNase digestion, lysates were loaded on a sucrose gradient and centrifuged at 100,000 rpm to recover ribosome protected fragments. RNA from the resuspended ribosomal pellet was purified and run on a gel to selectively excise footprinted RNAs (from 17 nt to 34 nt RNA fragments). To avoid any ribosomal contamination in the library preparation steps, we then performed Ribo-Zero Gold depletion of the footprinted RNA. The rRNA depleted RNA fragments were dephosphorylated and the linker was added. To specifically deplete unligated linker, yeast 5'-deadenylase and RecJ exonuclease digestion was performed library preparation steps were performed essentially as previously described: Linder, B. , Grozhik, A.V. et al. Nat methods 12, 767-772 (2015)
attributes.tag: GEO Accession
attributes.value: GSM2935911