GSM2580140: RNA-Seq HEK 293T; Homo sapiens; RNA-Seq
Total RNA from HEK 293T cells (N = 2 biological replicates) or whole mouse brain (N = 3 biological replicates per experimental group) was extracted using TRIzol (ThermoFisher) and treated with RQ1 RNase-free DNase I (Promega) according to the manufacturers’ instructions. Poly(A) RNA was then isolated using one round of selection with oligo(dT) magnetic beads (New England Biolabs). This resulted in approximately 10 μg of poly(A) RNA for each replicate used in this study. 1mA miCLIP was performed exactly as previously described(Linder et al., Nature Methods, 2015), with the following modifications: mRNA was subjected to fragmentation using RNA Fragmentation Reagents (ThermoFisher). Fragmentation was allowed to proceed for exactly 12 minutes at 75°C. Fragmented RNA was then incubated with anti-1mA antibody and the antibody-RNA complexes were processed for crosslinking, immunoprecipitation, RNA linker ligation, purification, and reverse transcription exactly as previously described(Linder et al., Nature Methods, 2015). For mouse brain samples, immunoprecipitated antibody-RNA complexes were mock-treated or treated with AlkB prior to RNA linker ligation. Reverse transcription incorporated use of barcoded primers to allow downstream pooling of libraries. Following reverse transcription of RNA-peptide complexes, first-strand cDNA was circularized using CircLigase II ssDNA Ligase (EpiBio) to preserve the 3’ end of the cDNA, and thus, sites of 1mA-induced terminations of reverse transcription. To generate priming sites for library amplification, the cDNA was cut in the middle of its priming sequence (green bar in Fig. 2a) using a small single-stranded oligo complementary to the priming sequence and FastDigest BamHI restriction digest (ThermoFisher), as previously described(Linder et al., Nature Methods, 2015). This generated priming sites for the Illumina P5 and P3 primers on either side of the cDNA. For library amplification, Accuprime Supermix I (ThermoFisher) and Illumina P5 and P3 primers were used. PCR amplification cycle number was selected as previously described(Grozhik et al., Methods in Molecular Biology, 2017). Amplified libraries were then pooled and cleaned up using a 1.8X ratio of AMPure XP magnetic beads (Beckman Coulter). Libraries were subjected to next-generation sequencing at the Epigenomics Core of Weill Cornell Medicine. Libraries were sequenced on an Illumina HiSeq 2500 instrument in single-end mode to generate 50-base reads.
GEO Accession
GSM2580140