GSM1854024: RNA Bisulphite sequencing WT2; Mus musculus; Bisulfite-Seq
Total RNA was extracted using Trizol reagents (Invitrogen) from snap‐frozen material and treated with DNase (Turbo DNase, Ambion). Ribosomal RNA was removed using Ribo‐zero (Epicentre, Illumina). tRNA sequencing library preparation: size‐selected or with the MirVana Isolation Kit (Invitrogen) for 20‐ to 200‐base‐paired RNAs. tRNAs were then de‐aminoacylated in 0.1 M Tris–HCl pH 9.0 and 1 mM EDTA for 30 min at 37°C. tRNA libraries were performed according to TruSeq Small RNA Preparation Kit (Illumina) instructions. Briefly, 3′ adenylated and 5′ phosphorylated adapters suitable for Illumina RNA sequencing were ligated to 300 ng of RNA obtained after de‐aminoacylation. RNA was reverse‐transcribed at 50°C for 1 h (SuperScript III cDNA synthesis kit, Invitrogen), followed by 16‐cycle PCR amplification with Phusion DNA polymerase (Thermo scientific) and size‐selected (140–210 base pairs) before sequencing. Bisulphite sequencing library preparation:  RNA was bisulphite‐converted as followed; about 2 μg of RNA was mixed in 70 μl of 40% sodium bisulphite pH 5.0 and DNA protection buffer (EpiTect Bisulfite Kit, Qiagen). The reaction mixture was incubated for four cycles of 5 min at 70°C followed by 1 h at 60°C and then desalted with Micro Bio‐spin 6 chromatography columns (Bio‐Rad). RNA was desulphonated by adding an equal volume of 1 M Tris (pH 9.0) to the reaction mixture and incubated for 1 h at 37°C, followed by ethanol precipitation. 120 ng of bisulphite‐converted RNA was used to generate Bisulfite‐seq libraries. Bs‐Seq libraries were performed according to TruSeq Small RNA preparation kit (Illumina). First 2′,3′‐cyclic phosphate and 5′‐hydroxyl termini produced during the bisulphite/desulphonation reaction were end‐repaired with T4 PNK and Spermidine (New England Biolabs). RNA adapters suitable for Illumina sequencing were then ligated, reverse‐transcribed at 50°C for 1 h with SuperScript III and 1 mM of each dNTP (SuperScript III cDNA synthesis kit, Invitrogen) followed by 18‐cycle PCR amplification. The amplified product was not size‐selected before sequencing. Small RNA Illumina library preparation protocols were used for both tRNA-seq and Bisulphite-seq.
GEO Accession
GSM1854024