title: GSM5259582: C1 Chemical Mapping - 1M7; synthetic construct; OTHER
library_construction_protocol: In vitro treated RNAs were purified by Zymo RNA Clean and Concentrator 5 kit. 10 µL of modified RNA was mixed with 1 µL of 200 ng/µL random nonamer primer (New England Biolabs), incubated at 65°C for 5 minutes, then snap-cooled on ice. Then, 8 µL of 2.5X MaP buffer (125 mM Tris pH 8.0, 187.5 mM KCl, 25 mM DTT, 1.25 mM each dNTPs, 15 mM MnCl2), was added to the primer-template mixture, incubated at room temperature for 2 minutes, and then mixed with 1 µL of SuperScript II (Invitrogen). The reverse transcription reaction was thoroughly mixed by pipetting, incubated at room temperature for 10 minutes, then at 42 °C for 3 hours. The reverse transcription enzyme was heat inactivated at 70°C for 15 min, snap-cooled on ice, then immediately mixed with 8 µL NEBNext Second Strand Synthesis Reaction Buffer (New England Biolabs), 4 µL NEBNext Second Strand Synthesis Enzyme Mix (New England Biolabs), and 48 µL water. The second strand synthesis reaction was mixed thoroughly through pipetting and incubated in a thermocycler with the heated lid off at 16°C for 60 min. The resulting double-stranded cDNA was purified with 1.8X volumes of Select-a-Size DNA Clean and Concentrator MagBeads (Zymo Research). 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)