GSM8067249: RPF-Vehicle-M1; Mus musculus; OTHER
The samples were lysed in cytoplasmic lysis buffer and clarified by centrifugation at 12,000 g for 10 min, Aliquots (100 and 200 μL) from each supernatant were generated. 100-µL aliquot of supernatant was used to extract total RNA for constructing RNA-seq libraries and 200-µL aliquot of supernatant was treated with nuclease provided by the TruSeq Ribo Profile Kit (illumina). Nuclease digestion was stopped by adding 15 µL of SUPERase-in (Thermo Fisher Scientific; AM2696). Size exclusion columns (illustra MicroSpin S-400 HR Columns) Size exclusion columns (illustra MicroSpin S-400 HR Columns; GE Healthcare; catalog no. 27-5140-01) were equilibrated with 3 mL of polysome buffer by gravity flow and spun at 600 × g for 4 min. Ribosomes were isolated by applying digested lysate immediately onto the prepared size exclusion columns above (100 µL of lysate per column) and spinning them at 600 × g for 2 min. Next, 10 μL 10% (wt/vol) SDS was added to the elution, and RNA with a size greater than 17 nt was isolated according to the Zymo RNA clean and concentrator kit (Zymo Research; R1017). After checking digestion quality, RNA with a size less than 200 nt was isolated according to the Zymo RNA clean and concentrator kit (Zymo Research; R1015). rRNA was depleted using the Ribo-Zero Human/Mouse/Rat kit (illumine; RS-122-2201, RS-122-2202, and RS-122-2203). After rRNA depletion, purified RNA was separated by 15% (wt/vol) TBE-urea PAGE (Thermo Fisher Scientific; EC68852BOX), and gel slices from 28 to 30 nt were excised. Ribosome footprints were recovered from the excised gel slices following the overnight elution method specified in the kit manual. After obtaining ribosome footprints above, Ribo-seq libraries were constructed according to TruSeq Ribo Profile kit manual and amplified by 13 cycles of PCR with a barcode incorporated in the primer. The PCR products were gel purified using the overnight method described by protocol. For RNA-seq, a 100-µL aliquot of supernatant as described above was used to extract total RNA by adding 5 µL of 10% (wt/vol) SDS followed by purification using the Zymo RNA clean and concentrator kit (Zymo Research; R1017). Then, 5 μg of total RNA were subjected to rRNA depletion using Ribo-Zero Human/Mouse/Rat kit (illumine; RS-122-2201, RS-122-2202, and RS-122-2203). The rRNA-depleted RNA was used to construct sequencing libraries using the TruSeq Ribo Profile kit (illumina). The circularized cDNA was amplified by 11 cycles of PCR and gel purified using the same procedure for the Ribo-seq libraries described above. Libraries were barcoded, pooled, and sequenced in a HiSeq 2500 machine (single-end 50 bp).