GSM8636677: Riboseq of mouse lung; Mus musculus; OTHER
Freshly collected mouse tissues were washed twice with cold PBS and the excess liquid was carefully aspirated. Then, 500 μl of lysis buffer (containing 8 mM HEPES, pH 7.4, 80 mM KCl, 4 mM MgCl2, 2% Triton X-100, and 200 μg/ml cycloheximide) was added to the tissues, which were thoroughly lysed. The lysate was then transferred to RNase-free tubes and centrifuged at 12,000 rpm for 12 minutes at 4°C. The supernatant was collected and sent to Novogene (China) for RNA-seq library construction. Additionally, approximately 100 μl of the supernatant was treated with RNase I (Thermo Fisher Scientific) for 3 hours, followed by RNA extraction using TRIzol™ LS. The extracted RNA was denatured at 65°C, 20 min and run on a 15% TBE-Urea gel. The band around 25-35 nt was excised and eluted overnight in RNA elution buffer (300 mM NaAc,1 mM EDTA) at 4°C. The RNA was precipitated with an equal volume of isopropanol. The RNA was treated by T4 Polynucleotide Kinase (NEB), and then polyadenylated using E. coli Poly(A) polymerase (NEB) for 37°C, 30min. The cDNA was generated using Template Switching RT Enzyme Mix from NEB (NEB) according to the manufacturer's instructions. A portion of the resulting cDNA was amplified by PCR using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) with barcoded primers. The PCR products were run on a 8% TBE gel, and the band around 180 bp was excised and recovered using DNA PAGE Gel Extraction Kit ( Beyotime). The recovered DNA was sent to Novogene for sequencing.