title: GSM5259587: P4P6 Chemical Mapping - NM; synthetic construct; OTHER
library_construction_protocol: In vitro treated RNAs were purified by Zymo RNA Clean and Concentrator 5 kit. 10 µL of the modified RNA was mixed with 1 µL of 10 µM oVT555 (GACGCCGTACGTACGCGG), incubated at 65 °C for 5 min, and snap cooled on ice. Then, the template-primer mix was combined with 4 µL of 5X TGIRT First Strand Synthesis Buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl, 15 mM MgCl2 ), 2 µL 10 mM dNTPs, 1 µL freshly prepared 100 mM DTT, and 0.5 µL TGIRT-III (InGex LLC). The reaction was mixed and incubated at 57 °C for 3 hours. RNA was then hydrolyzed by addition of 10 µL hydrolysis buffer (0.5 M NaOH, 0.25 M EDTA) and incubation at 65 °C for 15 min. Hydrolysis was quenched by bringing the reaction volume to 50 µL with water, adding 100 µL of Oligo Binding Buffer (Zymo Research), and proceeding through the Zymo Oligo Clean and Concentrator (Zymo Research) purification protocol, eluting in 15 µL of water. 5 µL of the purified cDNA was amplified in a NEBNext Q5 HotStart master mix PCR reaction containing 0.5 µM each oVT554 (GGGACATTTGCTTCTGACACAACTGTGTTCA) and oVT555 (GACGCCGTACGTACGCGG) with the following cycling conditions: 98 °C for 30 seconds, 10 cycles of 98 °C for 10 seconds followed by 72 °C for 60 seconds, with a final extension of 72 °C for 5 minutes. Products were purified using 0.9X Select-a-Size DNA Clean and Concentrator MagBeads (Zymo Research).Amplification of the full-length cDNA product was verified on an agarose gel stained with SYBRSafe (Invitrogen). Double-stranded cDNA was prepared for Illumina sequencing with the NEBNext Ultra II FS DNA Library Prep Kit for Illumina (New England Biolabs) and iTru primers. Briefly, 100-500ng of DNA from either 1M7 or DMS conditions in 26 µL of water was fragmented and end-repaired by adding 7 µL NEBNext Ultra II FS Reaction Buffer and 2 µL NEBNext Ultra II FS Enzyme Mix, mixed thoroughly by vortexing, and incubated in a thermocycler with heated lid on at 37°C for 20 min, then 65°C for 20 min. Fresh ligation adapter was prepared by denaturing a solution of 15 µM each of iTrusR1-stub (ACACTCTTTCCCTACACGACGCTCTTCCGATC*T) and iTrusR2-stubRCp (/5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTCA*C) in salty TLE (10 mM Tris pH 8.0, 0.1 mM EDTA, 100 mM NaCl) at 95°C for 1 minute, then annealed via slow cooling at -0.1°C/s to 25°C. The fragmented and end-repaired DNA was mixed with 2.5 µL 15 µM ligation adapter, 30 µL NEBNext Ultra II FS Ligation Master Mix, and 1 µL NEBNext Ultra II FS Ligation Enhancer. The ligation reaction was incubated in a thermocycler (heated lid off) at 20°C for 60 min. Ligated fragments were purified with 0.9X Select-a-Size DNA Clean and Concentrator MagBeads (Zymo Research) and eluted in 25 µL water. Dual-index sample barcodes were added through indexing PCR using the NEBNext Q5 Hot Start HiFi PCR Master Mix in a reaction with 0.5 µM each of iTru_5 and iTru_7 indexing primers with the following thermocycling parameters: 98°C for 30 s, 8 cycles of 98°C for 10 s, 55°C for 10 s, 72°C for 15 s, and a final extension at 72°C for 5 min. The final sequencing libraries were purified with 0.9X volumes of Select-a-Size DNA Clean and Concentrator MagBeads (Zymo Research)