GSM9291111: Ribo_hSLFN14_WT_-Dox; Homo sapiens; OTHER
WT or K219N SLFN14-inducible 293T cells were seeded in 6-well plates and left untreated or induced with doxycycline (2 μg/mL) for 24 h. The 6-well plates were pre-coated with poly-D-lysine (Gibco) for 5 min at room temperature to enhance the cell attachment. Following treatment, cells were washed once with ice-cold PBS and lysed in polysome lysis buffer (10 mM HEPES, pH 7.4, 5 mM MgCl₂, 100 mM KCl, 1% Triton X-100, 100 μg/mL cycloheximide). Lysates were clarified by centrifugation at maximum speed (≥ 13,000 × g) for 15 min at 4 °C, and the supernatant was collected for downstream ribo-seq analysis. Ribo-seq libraries were constructed by modifying the Ezra-seq method, as previously described. In brief, the whole cell lysates were digested with E. coli RNase I (Ambion, 750 U per 100 A260 units) by agitation at 4°C for 1 h. RNAs were extracted using Trizol LS reagent (Invitrogen) followed by isopropanol precipitation. The ribosome-protected mRNA fragments (RPFs) were separated on a 15% polyacrylamide TBE-urea gel (Invitrogen) and visualized using SYBR Gold (Invitrogen). Selected regions in the gel corresponding to 25-35 nt were excised and dissolved by soaking in 400 μl RNA elution buffer (300 mM NaOAc pH 5.2, 1 mM EDTA, 0.1 U/μl SUPERase·In (Invitrogen)) for 10 min at 70 °C. The gel debris was removed using a Spin-X column (Corning), followed by isopropanol precipitation. 4 μl RNAs (10~200 ng) were mixed with 1 nmol ATP, 1 μl T4 PNK (NEB), 20 U SUPERase·In, 1 μl Poly(A) Polymerase (NEB), and 1 μl homemade Ezra enzyme in 1 × T4 PNK buffer and incubated at 37 °C for 30 min followed by 70 °C for 10 min. Then ligation was performed for 90 min at 25 °C by mixing with a 10 μl reaction mixture (1 pmol biotinylated 5' end adaptor, 1 × T4 Rnl2 reaction buffer, 20 U SUPERase·In, 12.5% PEG8000 and 200 U T4 RNA ligase 2 truncated KQ (NEB)). The ligated RNA samples were pulled down after incubation with 20 uL pre-washed streptavidin magnetic beads (NEB) at room temperature for 10 min. After washing once with 100 uL 2 × SSC (Invitrogen), beads were resuspended in 12 μl nuclease-free water and mixed with 8 μl cDNA synthesis mixture (1 poml RT primer, 1 × first strand buffer, 100 nmol DTT, 1 nmol dNTP, 20 U SUPERase·In and 100 ng m-MLV) followed by incubation at 50 °C for 20 min. After washing once with 2 × SSC, beads were resuspended in 20 μl nuclease-free water and incubated at 95 °C for 2 min, then immediately placed on the ice for 1 min. After placing on magnet stand for 1 min, the supernatant cDNA was amplified by PCR using barcoded sequencing primers. PCR was performed by mixing 1 × HF buffer, 0.5 mM dNTP, 0.25 μM PCR primers and 0.025 U Phusion polymerase. PCR was carried out under the following conditions: 98 °C, 30 s; (98 °C, 5 s; 67 °C, 15 s; 72 °C, 20 s) for 13 cycles; 72 °C, 2 min. PCR products were separated on an 8% polyacrylamide TBE gel (Invitrogen). DNA products with the expected size 170 bp were excised and recovered from DNA elution buffer (300 mM NaCl, 1 mM EDTA). After quantification by Qubit 4 Fluorometer (Invitrogen), equal amounts of barcoded samples were pooled and sequenced using MiniSeq (Illumina).