IOX1 -- The Exhaustive Evaluation On What Works And What Doesn't

In brief, samples were taken from glucose-limited continuous cultures of E. coli K-12 BW25113 and its smpB deletion mutant14 in glucose MOPS medium after 30 s treatment with Cm I-BET-762 chemical structure (100 ��g/ml) or Tet (40 ��g/ml). RP following treatment with clindamycin (10 ?g/ml; clindamycin hydrochloride monohydrate, Tokyo Chemical Industry, Japan) and pactamycin (5 ?g/ml, Sigma) were similarly performed using an E. coli K-12 BW25113 tolC mutant,14 which is more sensitive to these antibiotics.19,20 Because the sequence libraries were constructed by adding polyA to the 3��-ends of the short RNA fragments produced with RNase I, the polyA sequences were computationally eliminated using fastx_clipper in the FASTX-Toolkit (http://hannonlab.cshl.edu/fastx_toolkit/index.html, accessed 27 February 2016) and mapped to the E. coli K-12 genome (GenBank: ""type"":""entrez-nucleotide"",""attrs"":""text"":""U00096.2"",""term_id"":""48994873"",""term_text"":""U00096.2""U00096.2) using bowtie.21 Number of reads mapped to coding region was about one to a few million (Supplementary Table S1). Mapped length of reads IOX1 distributes mainly from 25 to 50 nucleotides, due to size selection of digested mRNA fragments by polyacrylamide gel electrophoresis.11 The length distribution of TetRP reads mapped to CDS, indicating protected length by ribosome, is shown in Supplementary Fig. S1. To compare the read depth at each genomic position, the mapping information of each read was used to summarize the read depth at each position on both strands. For precise mapping, positions corresponding to 3�� ends of reads were used for calculation. The read depth was normalized between samples by defining the average depth over the entire coding region as one; the resultant depth was used as the signal strength at each position. Because 3�� adenines were removed during data processing, reads ending before and after a 3�� adenine were counted diglyceride as the same position, depth at this position was used for both positions. While this process reduced resolution, we found this step was a reasonable solution for purposes of data analysis.11 Though the protected length by ribosome ranges mainly from 25 to 50 nucleotides in the datasets, our previous analyses showed that it extends 12- to 13-nt in the 3��-direction from the first base of A-site codon but various lengths in the 5��-direction;11 thus, an average read depth over 15- to 16-nt 3�� was used as the signal to identify codons starting from a specific genome position. 2.3. Screening new translation start sites of known coding genes In-frame NTG codons