GSM4826367: shFBL_rep3_Ribo_seq; Homo sapiens; OTHER
Total RNA was isolated from cells using the RNA MiniPrep kit (Direct-zol, R2052, ZYMO Research) with the procedure provided by the manufacturer.  RNA libraries were prepared for sequencing using standard Illumina protocols Ribosome profiling experiment is performed following the method described in Methods. 2017 Aug 15;126:112-129.  Briefly, 4 million cells are rapidly harvested and lysed under conditions that maintain in vivo ribosome positions on mRNAs. These cell lysates are then subjected to nuclease footprinting with RNase I, and ribosomes are recovered by ultracentrifugation. Ribosome footprints are purified and ligated to a single-stranded linker. The RNA-DNA hybrid molecules are used as templates for reverse transcription, and following gel-based purification of full-length products. The first-strand reverse transcription products are then circularized. Finally, a double-stranded DNA library for Illumina sequencing is constructed from the single-stranded cDNA circles by means of a PCR amplification.