title: AF_45d_Ribo-seq
design: Briefly, skin tissues were lysed with polysome buffer [20 mM Tris-Cl [pH7.4], 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, 200g/ml cyclohexamide, 1 % (vol/vol) Triton X-100 and 25U/ml Turbo DNase I (Ambion, Cat# AM2238)] and lysate were digested with RNAse I (Ambion, Cat# AM2294). For RNAseq library construction, total RNA was extracted and oligo-dT magnetic beads were used to select RNA containing poly-A tails. Selected RNA was fragmented, and 50-60 nucleotide-long fragments were selected following end-repairing, linker ligation, reverse transcription, and circular PCR. Ribosome-protected fragments (RPF) purified with PAGE/fragmented total RNA were dephosphorylated with T4-PNK (NEB, Cat# M0201S). Linkers were ligated to RPF/fragmented total RNA by T4 Rnl2tr K227Q ligase (NEB, Cat# M0351L), and the cDNA synthesis was performed with SuperScriptIII (Invitrogen, Cat# 18080- 093). The cDNA samples were circularized by CircLigase (Epicentre, Cat# CL4111). The DNA libraries were amplified with Phusion High-Fidelity DNA Polymerase (NEB, Cat# M0530S) and purified with the AMPure XP system (Beckman Colter, Cat# A63880). The quantified libraries were sequenced with the Illumina sequencing platform following the manufacturers instructions.
library_name: 8