RPFs were isolated from an aliquot of frozen yeast lysate and sequenced on the Illumina HiSeq platform, as described (Subtelny et al., 2014 (link)). Detailed protocols for RNA-seq and ribosome profiling are available at http://bartellab.wi.mit.edu/protocols.html . RNase I treatment was performed using 0.2 U/µL lysate. Subtractive hybridization to remove contaminating rRNA fragments was performed using a mixture of three biotinylated oligonucleotides (Integrated DNA Technologies): 5'-GATCGGTCGATTGTGCACCTC/3Bio/; 5'-CGCTTCATTGAATAAGTAAAG/3Bio/; 5'-GACGCCTTATTCGTATCCATC/3Bio/
Biotinylated oligonucleotides
Manufactured by Integrated DNA Technologies
Biotinylated oligonucleotides are synthetic DNA or RNA sequences that have been modified by the addition of a biotin moiety. Biotin is a small molecule that can be used to facilitate the detection, immobilization, or purification of the oligonucleotide. The core function of biotinylated oligonucleotides is to enable these applications through the high-affinity binding interaction between biotin and streptavidin or avidin.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq platform, by Illumina (1 mentions)
Labchip gxii touch ht, by PerkinElmer (1 mentions)
Miseq instrument, by Illumina (1 mentions)
Kapa hyper prep kit, by Roche (1 mentions)
S220 focused ultrasonicator, by Covaris (1 mentions)
Seqcap ez system, by Roche (1 mentions)
Brightstar biodetect nonisotopic detection kit, by Thermo Fisher Scientific (1 mentions)
Northernmax kit, by Thermo Fisher Scientific (1 mentions)
Chemiluminescent nucleic acid detection module, by Thermo Fisher Scientific (1 mentions)
Tripure isolation reagent, by Roche (1 mentions)
5 protocols using biotinylated oligonucleotides
1
Yeast Ribosome Profiling and RNA-seq
2
Capture-C Library Preparation and Sequencing
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Capture-C was performed as described in (Davies et al., 2016 (link)) with some modifications. Capture probes were designed using CapSequm (Hughes et al., 2014 (link)). To prepare Capture-C libraries, 5 μg of 3C library were sonicated using a S220 focused ultrasonicator (Covaris) to 200 bp and 2.5 μg of fragmented DNA were processed with the KAPA Hyper Prep Kit (KK8500, Kapa Biosystems) according to manufacturer’s instructions. Two rounds of capture of respectively 72 and 24 hours were then performed, pooling 2 μg of each indexed library and using 13pmol of capture probes (biotinylated oligonucleotides, Integrated DNA Technologies), with the SeqCap EZ system (#06953212001, Roche/NimbleGen). This capture was performed according to manufacturer’s instructions, except for the first round when the volume of reagents was multiplied by the number of pooled libraries. Library size was confirmed using LabChip GXII Touch HT (Perkin Elmer) with a DNA High Sensitivity chip, and DNA concentrations were estimated using Qubit (Thermo Fisher Scientific). Capture-C libraries were sequenced on a MiSeq instrument (Illumina) using 75bp paired end reads and 5% PhiX.
3
Northern Analysis of PrrF and PrrH Small RNAs
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Northern analysis of the PrrF and PrrH sRNAs was performed as previously described with some modifications (Oglesby-Sherrouse and Vasil 2010 (link)). Briefly, 10–20 μg of total RNA isolated on RNeasy Mini Columns was run on a 6% polyacrylamide denaturing (7 mol/L urea) gel then transferred to a BrightStar membrane (Life Technologies, Grand Island, NY, USA) using a semi-dry transfer apparatus. Biotinylated oligonucleotides that were complementary to the regions of PrrF1, PrrF2, or PrrH as shown in Figure 1 A were purchased from Integrated DNA Technologies (IDT) and hybridized to blots overnight at 42°C. The membrane was washed using the Northern Max Low Stringency and High Stringency wash solutions according to the manufacturer's instructions. Detection of the biotinylated probes was carried out using the BrightStar BioDetect nonisotopic detection kit (Life Technologies).
4
Biotinylated Oligonucleotide Design
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Biotinylated oligonucleotides were ordered from Integrated DNA Technologies, using the TEG–biotin modification on the 5′ end of the oligo. All oligos were designed with Primer3 version 4.0 to be between 23 and 25 nucleotides in length, with a Tm as close to 63 °C as possible. Testing multiple oligonucleotides, it was found that those biotinylated oligos targeted to regions ∼150 bp from the targeted protein-binding site gave the most reliable data. The oligos were resuspended at 1 μg μl−1 in TE buffer and stored at −20 °C until use.
5
Analyzing mRNA Stability in E. coli
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E. coli cells were grown in a LB medium until the OD660 reached ~0.5. The expression of zntR mRNA or zntR‐arfA mRNA was then induced by 1 mM IPTG for 15 min. At this point, 200 μg/ml of rifampicin was added to the culture. The culture was harvested at 0, 1, 2, and 4 min after the addition of rifampicin. Total RNA was prepared using the Tripure Isolation Reagent (Roche), according to the supplier's instructions. RNA samples were separated by 1.5% denaturing agarose electrophoresis, transferred onto BrightStar‐Plus Positively Charged Nylon Membranes (Invitrogen), and hybridized with biotinylated oligonucleotides (Integrated DNA Technologies) complementary to the zntR or arfA mRNA, as shown in TableS4 . Hybridization experiments were performed using a NorthernMax kit (Ambion) and a Chemiluminescent Nucleic Acid Detection Module (ThermoFisher Scientific) according to the suppliers' instructions. Images were visualized and analyzed by a LAS4000 LuminoImager (Fujifilm).
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E. coli cells were grown in a LB medium until the OD660 reached ~0.5. The expression of zntR mRNA or zntR‐arfA mRNA was then induced by 1 mM IPTG for 15 min. At this point, 200 μg/ml of rifampicin was added to the culture. The culture was harvested at 0, 1, 2, and 4 min after the addition of rifampicin. Total RNA was prepared using the Tripure Isolation Reagent (Roche), according to the supplier's instructions. RNA samples were separated by 1.5% denaturing agarose electrophoresis, transferred onto BrightStar‐Plus Positively Charged Nylon Membranes (Invitrogen), and hybridized with biotinylated oligonucleotides (Integrated DNA Technologies) complementary to the zntR or arfA mRNA, as shown in Table
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