GSM1464623: CIRS-seq (Dimethyl sulfate, Replicate #1); Mus musculus; OTHER
DMS reaction was quenched by placing samples on ice and adding 0.7M final ice-cold 2-Mercaptoethanol and 1ml ice-cold TRIzol (Invitrogen). After chloroform addition and centrifugation, one volume of 100% ethanol was added to the upper aqueous phase, and sample was purified using RNEasy Mini Spin Columns (Qiagen) to allow complete removal of 2-Mercaptoethanol and DMS. CMCT reaction was stopped by addition of 1ml ice-cold TRIzol. Samples corresponding to 0mM DMS/CMCT (NT condition), were extracted in 1ml ice-cold TRIzol. For CIRS-seq library preparation, we first prepared pools of each treatment. The DMS sample was obtained by pooling the 50-100-150-200mM treated conditions, while the CMCT sample was obtained by pooling the 10-20-25-50mM treated conditions (~1.25 μg each). The samples obtained by treating with 0mM DMS and 0mM CMCT were pooled in equal amounts (~2.5 μg each), and constituted the Not treated (NT) control. Ribosomal RNA was depleted using Ribo-Zero Gold Kit (Epicentre). One-third of each sample (corresponding approximately to 100ng of Ribo- RNA) was subjected to reverse transcription with random hexamers using SuperScript II Reverse Transcriptase (Invitrogen). Reverse transcription reaction was carried out in 1 hour at 42°C, followed by 10 minutes at 70°C to inactivate the reverse transcriptase. Template RNA was then degraded by adding 10U of Ribonuclease H (Ambion) and incubating for 20 minutes at 37°C. After ethanol precipitation of cDNA, an adapter modified with a 5'-P group and a 3'-C3 spacer, corresponding to the reverse complement of the standard Illumina TruSeq Small RNA 5' adapter (RC5), was ligated to cDNA 3'-OH termini using 200U of CircLigase II for 4 hours at 68°C. This approach allowed us to keep the strand-specificity of the library, so that each read will start 1nt downstream of the RT stopping point. Then, to enable ligation of a 5' adapter, cDNA was treated with T4 Polynucleotide Kinase (NEB) in T4 DNA Ligase Buffer (NEB) for 1 hour at 37°C. After ethanol precipitation cDNA was loaded on a TBE-Urea 5% PAGE gel. A gel slice corresponding to 70-200nt was cut and cDNA was recovered by passive diffusion into diffusion buffer (500mM Ammonium acetate, 1mM EDTA, 10mM Magnesium acetate, 0.1% SDS) for 16 hours at 37°C, followed by ethanol precipitation. cDNA 5' termini was then ligated to a second adapter, corresponding to the reverse complement of the standard Illumina TruSeq Small RNA 3' adapter (RC3), using 200U of CircLigase II for 4 hours at 68°C. Adapter-ligated cDNA was then subjected to 15-18 cycles of PCR using standard Illumina TruSeq primers. To remove adapter dimers, library was loaded on a 3% (w/v) TBE-Agarose gel, and slice corresponding to 150-300nt was cut and purified using NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel).