GSM2454693: xzl.gga.WL.testis.wt.adult.rep1.RNAseq; Gallus gallus; RNA-Seq
Ribosome profiling Illumina-compatible sequencing libraries were prepared as the following.  The RPFs were then resolved on a 15% acrylamide (19:1), 8 M urea denaturing gel for 1 h 30 min at 35 W and fragments ranging from 26 nt to 35 nt size-selected from the gel. Size-selected RNAs were extracted from the gel slice by electro elution using GeBAflex tube, and the rRNAs were removed by Ribo-Zero Gold. The 3´ ends of recovered RNAs are dephosphorylated by PNK (New England BioLabs) in MES buffer (100 mM MES-NaOH, pH 5.5, 10 mM MgCl2, 10 mM ß-mercaptoethanol and 300 mM NaCl) at 37 °C for 3 h and then by Alkaline Phosphatase at 37 °C for 1 h. Dephosphorylated RNA footprints were then follow our small RNA library construction protocol except adding the step of 5´ end phosphorylation by PNK (New England BioLabs) using the PNK buffer with 1mM ATP at 37 °C for 1 h before 5´ ligation. Adaptor sequence: TGGAATTCTC. Strand-specific RNA-seq libraries were constructed following the TruSeq RNA sample preparation protocol as previously described (Li et al., 2013). rRNAs were depleted from total RNAs by Ribo-Zero Gold (Epicentre Biotechnologies, Madison, WI, USA). The library was sequenced using the paired-end 2 x 50 nt platform on a HiSeq 2000.