GSM7843970: Ribo footprints pTRIPZ RACK1, mutant RACK1 Replicate 2; Homo sapiens; OTHER
For performing ribosome profiling (Ribo-seq), ribosome-protected footprints were prepared for sequencing as described in a recently updated protocol of ribosome profiling (Chen et al., 2020; McGlincy and Ingolia, 2017).  Briefly, the cells were plated, grown, and subjected to the various treatments and experimental conditions indicated as described above.  They were the rapidly harvested and lysed.  Clarified cell lysates were treated with RNase I (Invitrogen) to digest RNA not protected by ribosomes.  The 80S ribosomes were isolated by centrifuging lysates through a 34% sucrose cushion at 100,000 x g for 1 hour at 4°C.  The RNA was purified from the ribosome pellet using the Direct-zol RNA kit (Zymo Research).  It was then resolved by electrophoresis through a denaturing gel, and the fragments corresponding to 28 to 34 bp were extracted from the gel. The 3' ends of the ribosome footprint RNA fragments were treated with T4 polynucleotide kinase (New England Biolabs, M0201) to allow ligation of a pre-adenylated DNA linker with T4 Rnl2(tr) K227Q (New England Biolabs, M0351S).  The DNA linker used incorporates sample barcodes to enable library multiplexing, as well as unique molecular identifiers (UMIs) to enable removal of duplicated sequences.  To separate ligated RNA fragments from unligated DNA linkers, 5'-deadenylase (Epicentre, DA11101K) was used to deadenylate the pre-adenylated linkers, which were then degraded by the 5'-3' ssDNA exonuclease RecJ (NEB, M0264S).  After rRNA reduction using the riboPOOL rRNA depletion kit (siTOOLs Biotech, H. sapiens pool), the RNA-DNA hybrid was used as a template for reverse transcription, followed by circularization with CircLigase (Epicentre, CL4111K). Finally, PCR of the cDNA circles was used to attach suitable adapters and indices for Illumina sequencing.