GSM3108530: 16_70S_EFG_UV_NAZ_SSII_Trunc; Escherichia coli; OTHER
Modified ribosomes were isopropanol precipitated, phenol extracted, reprecipitated. 1-4μg of total RNA was fragmented by incubation at 95°C for 5 minutes with 10mM ZnCl2. Fragmentation was stopped by placing tubes on ice and immediately adding EDTA to 20mM. RNA was ethanol precipitated, resuspended in 10mM tris pH 7.0 and run on a 10% PAGE TBE-urea gel for size selection. A section corresponding to 100-120bp on a DNA ladder was cut from the gel. The gel was shredded by forcing the gel through a needle hole in a 0.5ml tube by centrifugation, and RNA was eluted into 400μl water by heating at 70°C with rapid shaking for 15 minutes. Gel pieces were filtered out with a Spin-X column (corning) and RNA was isopropanol precipitated, resuspended in PNK mix (3.5μl 10mM Tris pH 8.0, 0.5μl SUPERaseIn RNase inhibitor (Invitrogen), 0.5μl 10x PNK buffer (NEB), 0.5μl T4 PNK), and incubated at 37°C for 1 hour. Reactions were supplemented with 5μl ligation mix (3.8ul 50% PEG 8000, 0.2ul 50μM pre-adenylated linker 2 (oBZ191) (For oligos, see supplementary Table S1), 0.5μl 10X T4 RNA ligase buffer (NEB), 0.5ul T4 RNA ligase 2 truncated (NEB)) and incubated at 37°C for 3 hours, then isopropanol precipitated. For E. coli experiments, pellets were resuspended in water and split for reverse transcription with either TGIRTII (Ingex) or SSII (Invitrogen). For all other experiments, only SSII was used, and pellets were directly resuspended in annealing mixture. For TGIRTII, 10ul reactions were prepared with RNA, 2μl Invitrogen 5x FS buffer, 0.5μl 10mM dNTPs, 0.5μl SUPERaseIn, 0.5μl 100mM DTT, .5μl 25μM RT primer oBZ192, 0.5ul 10μM TGIRTII, and incubated at 60°C for 1 hour. For SSII, 6μl annealing reactions were prepared containing RNA, 0.6μl 10x MapBasic Buffer (500mM Tris pH 8.0, 750mM KCl, 100mM DTT), 25μM RT primer oBZ192. RNA was annealed to primer by incubating: 4 minutes at 65°C, 2 minutes at 55°C, 2 minutes at 45°C, 2 minutes at 42°C, hold on ice. 6ul of extension mix (2.2μl water, 0.6μl MapBasic buffer, 0.6μl 10mM dNTPs, 0.5μl SUPERaseIn, 1.44μl 50mM MnCl2, 0.6μl SSII) was added, mixed, and incubated at 42°C for 1 hour. After all RT reactions, RNA was degraded by addition of 1μl 5M NaOH, and incubation for 3 minutes at 95°C. cDNA was precipitated, resuspended in 10mM Tris pH 7, and run on a 10% TBE-UREA gel. For mutation datasets, full length RT products were isolated. For RT stop datasets, cDNA was isolated from ~20nt above the RT primer to ~20nt below the full-length product. Gel extraction was performed as above, pellets were resuspended in Circ ligase mix (15.5μl 10mM Tris pH 8.0, 2μl 10x Circ ligase buffer, 1μl 1mM ATP, 1μl 50mM MnCl2, 0.5μl Circ Ligase I (Epicentre)), and incubated at 60°C for 2 hours, then 80°C for 20 minutes. Circularized cDNA was amplified with 8-12 cycles of PCR and subjected to Illumina sequencing on a HiSeq 2500 with 50bp single-end reads.
GEO Accession
GSM3108530