title: GSM8798514: hPSC_RiboSeq_Rep2; Homo sapiens; OTHER
library_name: GSM8798514
library_construction_protocol: RNA-seq: Total RNA was extracted from GFP+ and GFP- sorted samples in trizol using the DirectZol RNA Microprep kit (Zymo Research) following the manufacturer's instructions with on-column DNaseI treatment.    Ribo-seq: Ribosome footprinting was performed as described previously (Ingolia et al. 2012) with a few modifications. The GFP+ sorted cell pellets were lysed in buffer without cycloheximide. Following lysis and clarification by centrifugation, total RNA was quantified using the Qubit RNA BR assay kit. The remaining lysate was used as input into the footprinting reaction with 750 U RNase I (Invitrogen Ambion). The digestion was stopped with SUPERaseIN then flash frozen in liquid nitrogen. The lysate was then used as input into immunoprecipitation with RNA immunoprecipitation protocol adapted from (Wessels et al. 2016) using prepared Anti-FLAG M2 Magnetic beads (Sigma). The IP was incubated for two hours at room temperature tumbling end-over-end, followed by post-IP washes with NT2 buffer. The immunoprecipitated RNA was released from the bead complex using TRIzol LS reagent (Invitrogen) and further isolated using chloroform and isopropanol precipitation. RNA-seq: rRNA was depleted from each total RNA sample following our previously published RNaseH-based method (Baldwin et al. 2021), then input into the KAPA RNA Hyper prep RNAseq library prep kit (Roche). Libraries were prepared according to the 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 2x 150bp paired end sequencing on the Illumina NovaSeq6000 platform to a depth of 40 million paired end reads per library (University of Colorado Anschutz Genomics and Microarray Core Facility).        Ribo-seq: The isolated RNA was depleted of rRNA using the siTOOLs riboPOOLs kit for ribosome profiling following the kit instructions. The samples were resolved on a 15% TBE-Urea PAGE gel and ribosome protected fragments 28-29nt in size were extracted and recovered. RPFs were treated with T4-PNK then used as input into the Qiagen QIAseq miRNA library prep kit following the manufacturer's instructions with provided single index adapters. The resulting libraries were pooled and submitted for 2x 150bp paired end sequencing on the Illumina NovaSeq6000 platform to a depth of 60 million paired end reads (Supplemental Table 6) per library (University of Colorado Anschutz Genomics and Microarray Core Facility).
identifiers.namespace: GEO
identifiers.id: GSM8798514_r1