Unknown,Transcriptomics,Genomics,Proteomics

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RIL-seq analysis of MG1655 Escherichia coli experiencing long-term nitrogen starvation in Gutnick minimal media

ABSTRACT: RNA interaction by ligation and sequencing (RIL-seq) was carried out to compare the Hfq-RNA-RNA interactome of MG1655 E coli bacteria that grown in Gutnick minimal media. Wild-type and hfq::3xFLAG tagged K12 E.coli were grown in Gutnick minimal media with 3mM NH4Cl as the sole nitrogen source, and cell samples were taken during exponential growth (N+), ~20min following N-runout and induction of growth arrest (N-), 24hr following N-runout and induction of growth arrest (N-24) and 2hr following addition of NH4Cl to N-24 cells (N-24+2). All sampling was performed in triplicate for each strain and condition

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Escherichia coli K-12

SUBMITTER: Josh McQuail

PROVIDER: E-MTAB-13403 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Model topology is divided into two compartments, cell programming and performance testing. The cell programming compartment is split into history and pre-treatment. History ( History I or H1: E.coli grown for 18hrs in LB flask, transferred to fresh LB flask after that. History II or H2: E.coli grown for 18hrs in LB flask, transferred to fresh LB flask for 45 min. From this flask, 0.1O.D./ml transferred to rich medium and grown for 4 hours. From this,0.1O.D./ml transferred to fresh rich medium. History III or H3: E.coli grown for 18hrs in LB flask, transferred to fresh LB flask for 45 min. From this flask, 0.1O.D./ml transferred to starvation medium and grown for 4 hours. From this,0.1O.D./ml transferred to fresh starvation medium. Pre-treatment (Pre-treatment 1(T1): 2.5g glucose/litre 5mM NH4Cl. Pre-treatment 2 (T2): 2.5g glucose/litre 0.25mM NH4Cl. Pre-treatment 3 (T3): - 0.25g glucose/litre 5mmM NH4Cl. Pre-treatment 4 (T4): 0.25g glucose/litre 0.25mM NH4Cl). Each pre-treatment given for 2.5 hours.The culture nomenclature indicates the adaptive path followed, for example, H1T1 indicates the culture has encountered history I (H1) and then transferred to pre-treatment 1 (T1).RNA was extracted for selected combinations. Performance testing : Performance testing describes the type of analysis done which is the growth pattern study onto three substrates, glucose, succinate and pyruvate. This performance testing revealed specific history-pretreatment combinations to be better suited for growth on certain substrate and some not suited for growth. The samples were harvested for RNA isolation at peak growth points and named worst_glucose, best_glucose,worst_succinate, best_succinate, worst_pyruvate and best_pyruvate according to the growth shown after testing all 12 history-pretreatment combinations. The differences in physiology were studied in details using microarray analysis of 13 samples including 3 history samples, 4 pre-treatment samples and 6 samples at performance testing level. RNA extraction was done using Qiagen RNeasy minikit (Germany). Standard Affymetrix protocol was followed for hybridization on Affymetrix E. coli Genome 2.0 Array.

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RIL-seq analysis of MG1655 Escherichia coli experiencing long-term nitrogen starvation in Gutnick minimal media

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