Instrument: Illumina HiSeq 4000
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: For Ribo-Seq：5x10e6 DCs were treated with 100 μg/ml cycloheximide (CHX) for 7 min. The cells were then harvested by the cell lifter. The cell suspension was spun at 400g for 5 min and the cell pellet was washed twice with 5 ml cold PBS with CHX (100 μg/ml). 200 μl lysis buffer (10 mM Tris, pH 7.4, 150 mM KCl, 5 mM MgCl2, 100 μg/ml CHX, 0.5% Triton-X-100, freshly add 1:100 protease inhibitor, 40 U/ml SUPERasin) was added to the cell pellet and lysed on ice for 15 min with rotating; For m6A-Seq: Total RNA was isolated from DCs. Polyadenylated RNA was further enriched from total RNA by using Dynabeads® mRNA Purification Kit (Invitrogen). For Ribo-Seq：10% clarified lysate was saved as INPUT and the rest lysate was separated through a 5ml 10%-50% sucrose gradient and centrifuged at 4°C for 2 h at 28,000 r.p.m. Fractions were collected separately and analyzed by Qubit™ RNA HS Assay Kit (Invitrogen). The fractions corresponding to ribosomes were combined and concentrated on Amicon-Ultra 100K columns (Millipore). 2 A260 units of ribosome fractions were digested with 60U RNase I (Ambion) at room temperature for 30 min. RNA was extracted by RNA Clean&Concentrate (Zymo) and ribosomal RNA were deleted prior to size selection. RNA fragments (26-32nt) were isolated by 15% denaturing Urea-PAGE gel. RNA was eluted from gel in elution buffer (300 mM sodium acetate pH 5.2, 1 mM EDTA) followed by phenol-chloroform extract and ethanol precipitation. RNA fragments were dephosphorylated and prepared into libraries by SMARTer® smRNA-Seq Kit (Clontech); For m6A-Seq: RNA samples were fragmented into ~100-nucleotide-long fragments with sonication. Fragmented RNA (100ng mRNA or 5µg total RNA) was performed m6A-IP following EpiMark N6-methyladenosine enrichment kit (NEB E1610S) protocol. RNA was enriched through RNA Clean&Concentration-5 (Zymo Research) and used for library generation with SMARTer Stranded Total RNA-Seq Kit (Takara).