GSM8991824: Ribosome profiling, rep2,read1; Homo sapiens; OTHER
Four replicates of ~8 X10^6 HEK293T cells were plated in 150 cm2 plates. After ~18 h, cells were washed and then scraped with ice cold PBS, followed by centrifugation at 300 g for 5 min. 600 ul Lysis buffer (20 mM Tris, 150 mM NaCl, 5 mM MgCl2, 1mM DTT, Triton and 100 ug/ml of cycloheximide) was added to the cell pellet followed by incubation on ice for 10 min and the lysate was clarified by centrifugation at 1300g for 10 min at 4Â°C. The cleared lysate (50 ul) was directly used for total RNA extraction by adding 700 ul of Trizol (Zymo). 200 ul of the lysate along with 20 ul of the Ribonucleoside Vanadyl Complex was layered on the 1M sucrose cushion and subjected to ultracentrifugation at 38000 rpm for 2.5 h at 4°C. The remaining 350 ul was used for ribosome profiling. The pellet was dislodged in 700 ul of Qiazol and stored -80 °C until use the next day. Total RNA and translating RNA was extracted using Zymo RNA miniprep kit as per manufacturer's protocol (Zymo). The concentration was estimated using nanodrop and the integrity of the extracted RNA was determined by 2100 Bioanalyzer (RNA 6000 Pico Kit, Agilent). 1μg of the extracted RNA was used for Poly A selection using NEBNext poly(A) mRNA magnetic isolation kit (NEB, E7490S). This was followed by library preparation using NEBNext Ultra II RNA library prep kit for Illumina (NEB, E7770S) as per the manufacturer's protocol using 7 PCR cycles. The quality and concentration of the libraries formed were determined by Bioanlyzer (High sensitivity DNA assay kit, Agilent). Equimolar amounts of the libraries were pooled and sequencing was done using NovaSeqx Plus (2X150 paired end). For ribosome profiling, RNA was isolated using miRNeasy Mini kit and size-selected by running 1 µg of each sample in a 15% polyacrylamide TBE-UREA gel. 26–34 nt RNA fragments were excised, RNA fragments were extracted and precipitated. Ribosome profiling libraries were prepared using the D-Plex Small RNA-Seq Kit (Diagenode) as previously described. The cDNA was amplified for eleven cycles. Each library was cleaned with AMPure XP beads and eluted with 30 μL of RNase-free water. To completely remove the primer-dimers, size-selection was done using a 12 % polyacrylamide gel followed by excision of the fragment (~200bp). The DNA was extracted by overnight incubation in a buffer (10 mM magnesium acetate tetrahydrate, 0.5 M ammonium acetate, 1 mM EDTA (pH 8.0) at room temperature followed by ethanol precipitation (-20 °C, overnight) to recover the size selected libraries. The resulting libraries were analyzed with the Agilent High Sensitivity DNA Kit (Agilent). Each library was mixed in equimolar proportion for sequencing with the NovaSeqx Plus (2X150 paired end).