Genome-wide ribosome profiling to identify regulatory targets of small RNA RyhB in E.coli
Genome-wide ribosome profiling to identify regulatory targets of small RNA RyhB in E.coli
200 ml LB medium was inoculated with 2.0 ml of overnight E.coli culture, and grew at 37 degrees C to an OD600 of ~0.5. Arabinose was added to 0.1% 10 minutes before harvesting the cells to induce the expression of RyhB. Then cells were harvested by pretreatment with chloramphenicol to a final concentration of 100 μg/ml for 2 minutes followed by rapid filtration by using a 500ml 0.45um PES filter system (Celltreat). Cells were collected and flash frozen in liquid nitrogen together with 0.65 ml lysis buffer (Oh et al. 2011). The frozen cells were pulverized 6 times at 15 Hz for 3 min by a mixer mill (Retsch MM400). The grinding jars were re-chilled in liquid nitrogen to keep the cells frozen between each cycle. After the pulverized cells were recovered, small scoopful was saved for bacterial transcript enrichment. The pulverized cell powder was extracted with acid phenol and chloroform followed by isopropanol precipitation as described (Oh et al. 2011). 16S and 23S ribosomal RNAs were removed by subtractive hybridization using Ambion MICROExpressTM kit following manufacturer's manual. The enriched mRNAs were randomly fragmented as described (Oh et al. 2011) Both ribosomal footprints and total mRNAs were converted into cDNA libraries as described (Ingolia 2010, Oh et al. 2011) with modification. The RNA molecules were dephosphorylated by treating with T4 polynucleotide kinase (New England Biolabs). Then polyacrylamide gel purification was performed for size selection of ~28nt RNA fragments. Approximately 25-30 nt poly-A tails were added to recovered RNA fragments with an Ambion poly(A) tailing kit (Life Technologies)  following the manufacturer's guide. The polyadenylated RNA samples were reverse transcribed using JW2364 and SuperScript III (Life Technologies), and the reverse transcription products were circularized by CircLigaseTM ssDNA ligase (Epicentre). For the footprint libraries, ribosomal RNAs were subtracted from circularized ssDNA using biotinylated sense-strand oligonucleotides JW2808, JW2809, JW2810, and JW2811 as described (Oh et al. 2011). PCR amplification was performed by using the circularized cDNA as template, JW2365 and JW2366 (Table 1) as primers, and Phusion High-Fidelity DNA Polymerase (New England Biolabs).
Experimental Factor: genotype
No RyhB expression
Experimental Factor: experiment type
RNA-seq