GSM9267405: DAMER_M2, without ATF4, 1M7; Homo sapiens; OTHER
500 ng of in vitro transcribed DAMER and/or 500 ng of ATF4 mRNA were used.RNA was denatured at 95°C for 2 minutes and snap-cooled on ice.RNA was then refolded by incubating at 37°C for 20 minutes in 3.3× folding buffer (333 mM HEPES pH 8.0, 333 mM KCl, 33.3 mM MgCl2).Folded RNAs were treated with 100 nM of the SHAPE reagent 1-methyl-7-nitroisatoicanhydride (1M7) (as a negative control) for 5 minutes at 37°C. The modified RNA was purified using RNeasy Mini columns (Qiagen).Reverse transcription was performed using SuperScript II Reverse Transcriptase with specific primers in MaP buffer. Sequencing libraries were generated from the resulting cDNA using the Nextera XT DNA Library Preparation Kit (Illumina). Sequencing was performed on a NovaSeq 6000 system with PE250 reads.