GSM9130088: NC,UPF1,10-3; Neurospora crassa; OTHER
total RNA was extracted with Trizol reagent. 1.For SLAM-seq :After alkylation modification with iodoacetamide, 1ug total RNA (HCT116 cell) or 50 ng mRNA (N. crassa) was prepared for library. and library preperation was following the manufacturers protocal of VAHTS Universal V8 RNA-seq Library Prep Kit 2.For Ribo-seq in human cell : 2×10? HCT116 cells were lysed in 300 µL ice-cold lysis buffer (20 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl?, 1 mM DTT, 100 ?g/mL cycloheximide, 0.5% Triton X-100, 2 U/mL DNase I, 1× protease inhibitor cocktail). Lysates were centrifuged at 10,000 × g for 10 min at 4°C. Supernatants were treated with RNase I (1 U per OD??? unit) for 45 min at 25°C. Total RNA was extracted using TRIzol reagent, precipitated with glycogen, and washed with 80% ethanol. Ribosome-protected fragments (28-34 nt) were resolved on 16% TBE-urea PAGE, excised, and eluted in RNA Gel Extraction Buffer (300 mM NaOAc, 1mM EDTA, 0.25% SDS). RNA was precipitated with 1× volume isopropanol at -20°C overnight. For library preparation, 100-200 ng RNA fragments were adenylated using poly(A) polymerase and PNK (37°C, 30 min). Preadenylated 5' adapter were ligated to ribosome-protected fragments with T4 RNA Ligase 2, truncated KQ (25°C, 2 h). Then purifying the ligation product with Streptavidin magnetic bead. And template-switching primer was used in reverse transcription with SuperScript III reverse (50°C, 30 min), following the library amplification.