GSM3081752: LC502_normal_RPF; Homo sapiens; OTHER
Tumors and adjacent normal tissues from HCC patients were flash-frozen and stored in liquid nitrogen. Prior to the experiments, the tissue samples were crushed into powder in an automatic cryogenic grinder (HORIBA, Freezer/Mill 6770) filled with liquid nitrogen. About 50 mg of each of the powdered samples was lysed in 400 ul of lysis buffer containing 20 mM Tris-HCl (PH 7.4) (Invitrogen, AM9850G), 150mM NaCl (Invitrogen, AM9760G), 5mM MgCl2 (Invitrogen, AM9530G), 1% Triton X-100 (Sigma Aldrich, T8787), 0.1% NP40 (Amresco, E109), 1 mM DTT (Sigma Aldrich, 43816), 25 U/ml DNase I (Invitrogen, AM2239) and 0.1 mg/ml cycloheximide (Inalco, 1758-9310). The mixture was clarified for 10 minutes at 20,000 g at 4°C. 200ul of the clarified lysate was used for purification of the RPF, which will be profiled by ribosome profiling, and 100ul of the lysate was for the total RNA sequencing in parallel. For RPF, 90 Units of RNase I (Life Technologies, AM2294) for each A260 of the lysate was added to 200 ul of the supernatant and incubated at room temperature for 45 min with gentle mixing. Nuclease digestion was stopped with 15 ul of SUPERase InTM RNase Inhibitor (Life Technologies, AM2696) and chilled on ice. Ribosome-RNA complexes were purified by Sephacryl S400 spin column chromatography (GE Healthcare, 27514001). The ribosome-protected RNA was extracted with Trizol (Invitrogen, 15596018), following the manufacturer's protocol. Then the ribosomal RNA was depleted using the RiboZero kit (Illumina; MRZH11124), following the manufacturer's protocol, omitting the 50°C incubation step. Next, the samples were resolved in a 15% Urea gel by electrophoresis, and the fragment at 25-35 nucleotides was excised from the gel. The RNA fragments were then eluted at least 2h in 400ul Nuclease-Free Water, 40 ul of 5 M Ammonium Acetate (Invitrogen, AM9070G) and 2 ul of 10% SDS (Invitrogen, AM9823), followed by Isopropanol (Sigma Aldrich, I9030) precipitation. Total RNA from the tissue samples was isolated from 100 ul of the clarified tissue lysate with Trizol (Invitrogen, 15596018), and the ribosomal RNA was then depleted using the RiboZero kit (Illumina; MRZH11124). Next, the rRNA-depleted total RNA was fragmented with PNK buffer (NEB, M0201L) at 95˚C for 20min. Both RPF and fragmented total RNA were cloned and amplified for next-generation sequencing with a tagging-based workflow. In brief, the RNA fragments were end repaired with T4 PNK (NEB, M0201L) and ligated with 3' adaptor, followed by cDNA synthesis, cDNA gel purification, circularization and PCR amplification20. The sequencing libraries were assessed with a BioAnalyzer and quantified using KAPA SYBR FAST Universal qPCR Kit (Kapa Biosystems, KK4601) prior to and after pooling for sequencing. Library insert sizes were typically about 30bp. The pooled libraries were sequenced on the Illumina HiSeq 2500 platform with a single-end sequencing strategy for 50 cycles.
GEO Accession
GSM3081752