GSM8386315: Spinach, - Reduction + Depurination, 0 mM, SSII; synthetic construct; OTHER
In vitro transcribed RNA was purified from the quenched DMS reaction with RNA Clean and Concentrator 5 kit and eluted in 10µL water. RNA was either directly processed or stored at -80°C We noticed that DMS probing caused a large portion of adherent cells to become detached from the plate. We therefore decanted the quenched DMS probing media off the plate into a 15 mL falcon tube and pelleted the detached cells for 5 min at 4C and 1,000 x g. To isolate the total RNA, 2 mL Trizol reagent was added to the plate for the cells which had not become detached. Then, the 2 mL Trizol reagent was transferred to solubilize the pelleted cells which had become detached during DMS probing. Total RNA was then isolated according to manufacturer's instructions. Sequencing library preparation: Reverse transcription Superscript II: Reverse transcription with SSII was performed as follows. 200 ng of input RNA was mixed with 1µL 50µM random hexamer (HeLa total RNA) or 2µL of 1µM gene specific reverse transcription primer (in vitro transcribed RNAs) and annealed. Annealed RNA was added to reverse transcription master mix containing 6 mM final concentration of MnCl2. Reverse transcription reactions were incubated at 23°C for 15 min only when using random hexamers then 42°C for 3 hours. Marathon RT: Reverse transcription with Marathon RT was performed with a modified protocol. First, 200 ng of input RNA was mixed with 2µL10 mM dNTP and 1µL 50µM random hexamer or 2µL of 1µM gene specific RT primer. The volume was brough up to 8µL and the RNA was annealed to the primer with the following thermocycler settings: 80°C for 1 min, 65°C for 5 min at which point the RNA was placed on ice for 2 min. Then, an RT master mix was created containing 4µL of 5x Marathon MaP buffer (250 mM Tris-HCl pH 7.5, 1 M NaCl), 4µLof 100% glycerol, 1µL of 100 mM DTT, 0.5µL of 20 mM MnCl2, 0.5µL of water. Next, 10µL of RT master mix was added to 8µL of annealed RNA followed by the addition of 2µL Marathon RT. Reverse transcription reactions were incubated at 23°C for 15 min only when using random hexamers then 42°C for 3 hours. Reverse transcription reactions were inactivated by incubating at 75°C for 10 min. cDNA was purified with DNA clean and concentrator 5 columns by using 7 volumes of DNA binding buffer (140 µL) and eluted in 10µL water. Sequencing libraries were generated via a 2 step PCR. Briefly, 1/5th of purified cDNA (2 µL) was amplified with step 1 forward and reverse primers. HeLa 18s rRNA samples included a 4-nucleotide barcode on the forward primer and a 7-nucleotide barcode on the reverse primer which enabled pooling of purified step 1 PCR products. AKT2 step 1 PCR primers were designed to amplify a ∼200 nucleotide segment of the AKT2 3'UTR. In vitro transcribed RNAs used non-barcoded step 1 forward and reverse primers which were specific to flanking sequences introduced into the RNA constructs. These flanking sequences are referred to as a Structure Cassette (SC). Step 1 PCR reactions were assembled on ice by adding to 2µL purified cDNA: 1.25µL of 10µM forward primer, 1.25µL of 10µM reverse primer, 9µL of water and 12.5µL of 2x Phusion HF master mix or 2x Phusion GC master mix for AKT2 step 1 PCR. Step 1 PCR reactions were run with the following thermocycler settings: 98°C for 2 min followed by 8-28 cycles of 98°C for 10 seconds, 65°C for 20 seconds, 72°C for 20 seconds, followed by a final extension of 2 min. PCR cycle number was optimized for each experiment separately. PCR reactions were purified with 1.8x AMPure XP beads according to manufactures instructions. Purified DNA was eluted in 25µL water. Step 2 PCR was performed with NEB Next Multiplex Oligos for Illumina. Step 2 PCR reactions were composed of the following: 2-10 ng of purified step 1 DNA brough up to 12µL water, 4µL of i5 index primer or universal primer, 4µL i7 index primer, and 20µL of 2x Phusion HF or 2x Phusion GC for AKT2 library prep. Step 2 PCR reactions were run with the following thermocycler settings: 98°C for 2 min followed by 6-12 cycles of 98°C for 15 seconds, 65°C for 30 seconds, 72°C for 30 seconds, then 72°C for 5 min. PCR cycle number was optimized for each experiment separately. Step 2 PCR reactions were size selected with AMPure XP beads, eluted in 25µL of water, and quantified with Qubit 1x dsDNA HS kit. Libraries were pooled and sequenced on the NovaSeq 6000 PE 2x100, NovaSeq 6000 PE 2x150, MiSeq PE 2x150, Nextseq2000 P1 600 cycles, or MiSeq Micro 300 cycles. Gene-specific BASH MaP: Total RNA was treated with DNAse I for 30 min prior to BASH MaP treatment according to manufacturer's instructions. Three replicates of 1 µg of total RNA was mixed with 1µL 50µM random hexamers and the volume was adjusted to 11 µL. RNA was annealed by heating to 85°C for 1 min, 65°C for 5 min, then placing on ice for 3 min. Reverse transcription was performed with SuperScript II as described previously8. Reverse transcription reactions were incubated at 23°C for 15 min and then 42°C for 3 hours. Reactions were inactivated by heating to 75°C for 10 min. Reverse transcription replicates were pooled and the cDNA was purified with the Zymo DNA Clean and Concentrator 5 kit and eluted in 10µL water.