GSM8198206: parental_ribo_a.1; Homo sapiens; OTHER
For RNA-seq RNA was isolated using Trizol LS and ZYMO Directzol RNA microprep kit with on-column DNase I treatment. PolyA(+) RNA was selected from 500 ng of each input sample using the NEBNext Poly(A) mRNA magnetic isolation module. For ribo-seq cells were lysed in lysis buffer containing 1ug/mL cycloheximide. Lysates were treated  with 750 U RNase I (Invitrogen Ambion). The digestion was stopped with SUPERaseIN followed by recovery of monosomes with equilibrated Microspin S-400 HR (GE) columns. Trizol LS was added to the monosome-containing elution and RNA isolated with the ZYMO Directzol microprep kit with on-column DNase I treatment. The isolated RNA was depleted of rRNA using the siTOOls riboPOOLs kit for ribosome profiling. The samples were resolved on a 15% TBE-Urea PAGE gel and ribosome protected fragments 28-29 nt in size were extracted and recovered. RPFs were treated with T4-PNK. RNA-seq libraries were prepared using KAPA RNA Hyper prep RNAseq library prep kit (Roche) with with dual-index adapters, followed by quality check of the final libraries by Qubit dsDNA HS assay and Tapestation High Sensitivity D1000 ScreenTape. Ribo-seq libraries were prepared using Qiagen QIAseq miRNA library prep kit with provided single index adapters.