GSM9456627: mdamb231_rna_si3e_normoxia_b; Homo sapiens; RNA-Seq
Ribosome profiling was performed as described previously with a few modifications (Calviello et al, 2016). The lysis buffer contained 100 ug/mL cycloheximide. Following lysis and clarification by centrifugation, total RNA was quantified using the Qubit RNA BR assay kit. A portion of the lysate was reserved as input sample for RNASeq preparation. Total RNA was extracted using the DirectZol RNA Microprep kit (ZYMO Research) following the manufacturer's instructions with on-column DNase I treatment.  Ribosomal RNA was depleted from total RNA using our previously described RNaseH-based method (Baldwin et al, 2021), then input into the KAPA RNA Hyper prep RNAseq library prep kit (Roche) according to manufacturer's instructions with dual-index adapters, followed by quality check of the final libraries by Qubit dsDNA HS assay and Tapestation High Sensitivity D1000 screentape. Libraries were pooled and submitted for paired-end 150 bp sequencing on the Illumina NovaSeq6000 platform to a depth of 40 million paired end reads per library. The remaining lysate was used as input into the ribosome footprinting reaction 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 (RPFs) 28-29 nt in size were extracted and recovered. RPFs were treated with T4-PNK then used as input into the Qiagen QIAseq miRNA library prep kit with provided single index adapters. Libraries were pooled and submitted for paired-end 150 bp sequencing on the Illumina NovaSeq 6000 S4 platform to a depth of 50 million paired end reads per library (Novogene Corporation Inc.).