Name: GSM5885021
Instrument: NextSeq 550
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Cell extract was treated with MNase, CaCl2 (5mM final concentration), and Turbo DNAse I and incubated at 25°C for 30 minutes; the digestion was stopped with SUPERase-inhibitor and placing on ice. The cell extract was loaded onto a 15%-45% sucrose gradient to separate the polysomes by ultra-centrifugation. The relevant fractions corresponding to the 80S monosome were collected and precipitated using ethanol and the RNA was isolated Total RNA was isolated from each sample and subjected to the Turbo DNase I digestion and then RNA-seq libraries. The ribosome-protected fragments were subjected to denaturing polyacrylamide-mediated gel electrophoresis and were gel-extracted based on size (26-mers to 34-mers) followed by rRNA depletion by RiboMinus kit (Ambion) For total RNA sequencing, libraries were constructed using Illumina's TruSeq Stranded Total RNA kit. The ribosome protected fragment library was generated using NEXT-Flex smRNA-Seq Kit v3 (Perkin Elmer)