title: GSM3067420: WT RPF; Homo sapiens; RNA-Seq
library_construction_protocol: Ribosome profiling libraries from 3 pooled cell culture plates were prepared using a standard protocol (McGlincy and Ingolia, Methods 2017), with minor modifications. Separate 5' and 3' linkers were ligated to the RNA-fragment instead of 3' linker followed by circularization (Subtelny et al, Nature 2014). 5' linkers contained 4 random nt unique molecular identifier (UMI) similar to a 5 nt UMI in 3' linkers. During size-selection, we restricted the footprint lengths to 18-34 nts. Matched RNA-seq libraries were prepared using RNA that was randomly fragmentation by incubating for 15 min at 95C with in 1 mM EDTA, 6 mM Na2CO3, 44 mM NaHCO3, pH 9.3. RNA-seq fragments were restricted to 18-50 nts. Ribosomal rRNA were removed from pooled RNA-seq and footprinting samples using RiboZero (Epicenter MRZH116). cDNA for the pooled library were PCR amplified for 15 cycles.