Instrument: Illumina HiSeq 4000
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: Ribosome profiling libraries were prepared using the TruSeq Ribo Profile (Mammalian) Kit (Illumina, San Diego, CA) with the following adjustments: Flash-frozen cortex samples were homogenized in lysis buffer including cycloheximide Keeping 1/5 of the supernatent aside for input libraris, 4/5 of the supernatent were digested with nuclease for ribosome profiling and purified on a sucrose gradient. Trizol LS purified ribosome protected fragments were processed as described in the TruSeq Ribo Profile Kit with rRNA depletion by RiboZero Gold (mammalian) and remaining fragments size-selected, purified and processed as described. cDNA was purified using a 2.5x AMPure clean-up. PCR was performed on undiluted circularized cDNA with 12 PCR cycles and PCR products on DNA-TBE PAGE. Sequencing was performed on each one lane for ribosome bound fractions and input fractions (indexed each 1-12) of an Illumina HiSeq4000 PE 2x100 (Illumina, San Diego, CA) using only the reverse read (R2).