GSM5699603: PLATE_ERCC_pool; Homo sapiens; OTHER
PLATE-seq: Following lysis in a polysome-preserving buffer containing RNase inhibitors, cycloheximide, and magnesium, all polyadenylated RNA was isolated by pulldown on a poly(T) capture plate. riboPLATE-seq: Following lysis in polysome-preserving buffer containing RNase inhibitors, cycloheximide, and magnesium, ribosome-associated RNA was isolated by indirect IP, in which lysates were combined with streptavidin-coated magnetic beads following a pre-incubation with biotinylated y10b anti-rRNA antibody. Next, the polyadenylated subset of this ribosome-associated RNA was isolated with use of a poly(T) capture plate. Ribosome Profiling: Monosomes were isolated from RNase I-digested TS543 lysates on a sucrose gradient fractionator. RNA-Seq: Total RNA was isolated from TS543 lysates using a Qiagen RNeasy kit (cat no. 74104) and ribosomal RNA was depleted using the Ribo-Zero rRNA removal kit from Illumina (Cat no. MRZH11124) according to the manufacturer's instructions. PLATE-seq/riboPLATE-seq: Polyadenylated RNA (for PLATE-seq) or ribosome-associated polyA RNA (for riboPLATE-seq) was barcoded through reverse transcription primed with barcoded, adapter-linked oligo(dT), after which single-strand cDNA were pooled. Second-strand synthesis with adapter-linked random hexamers, cDNA purification, and PCR enrichment were all performed on these 96-way multiplexed pools with the Hamilton Microlab STAR liquid handling robot according to the PLATE-seq protocol (Bush et al, 2017). Ribosome profiling : Conventional ribosome profiling libraries were prepared from size-selected ribosome footprints following the protocol given in (Ingolia et al., 2012), with minor modifications. Ligation-free ribosome profiling : Ligation-free ribosome profiling libraries were prepared from dephosphorylated foot-prints (~ 28-34 nucleotides in length) using a commercially available kit (SMARTer small RNA-Seq Library Preparation Kit, Clontech, Cat no. 635029) following manufacturer's instructions.   RNA-Seq : rRNA depleted total RNA samples were converted to  strand-specific sequencing libraries using the NEBNext® Ultra™ Directional RNA Library Prep Kit from Illumina (Cat no.E7420S).