GSM3994955: RNase P and tmRNA in-cell minusDMS; Escherichia coli; OTHER
Reverse-transcription was performed using specific primers, and DNA libraries were generated using a two-step PCR protocol. Sample-specific modification and extraction details are provided in the SAMPLES section. All samples were DNase treated and purified using ethanol precipitation prior to reverse transcription. Mutational profiling (MaP) reverse transcription (RT) was performed using an updated protocol that substantially improves yield and product length. In a 10 μL volume, RNA was mixed with 200 nM specific primer or 20 ng/μL random 9mer and 2 mM dNTPs and incubated at 65 °C for 10 min followed by 4 °C for 2 min. 9 uL 2.22x MaP buffer [1x MaP buffer consists of 6 mM MnCl2, 1 M betaine, 50 mM Tris (pH 8.0), 75 mM KCl, 10 mM DTT] was added and the combined solution incubated at 23 °C for 2 min. Finally, 1 μL SuperScript II Reverse Transcriptase (Invitrogen) was added and the RT reaction was performed according to the following temperature program: 25 °C for 10 min, 42 °C for 90 min, 10x[50 °C for 2 min,  42 °C for 2 min], 72 °C for 10 min. For the adenine riboswitch, ~1 pmol RNA was input into RT. For E. coli and human short RNAs, 0.5-2 μg total RNA was input into RT. For randomly primed E. coli RNA samples (16S and 23S rRNA), 1.5-3 μg total RNA was input into RT. RT products were then purified (Agencourt RNAclean XP beads, Beckman Coulter). Sequencing adaptors were added according to protocols listed in the SAMPLES section.