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Sample GSM3681087

Query DataSets for GSM3681087

Status

Public on Sep 20, 2019

Title

pair1_50mM_Mg

Sample type

SRA

Source name

pair1_50mM_Mg

Organism

Escherichia coli str. K-12 substr. MG1655

Characteristics

interface mutant: mutant pair 1 - PhoQ(TLSLKA)/PhoP(TPLRH)
mgso4 treatment: 50mM
trans-zeatin treatment: 0mM
genotype/variation: deltaphoPQ deltalacZYA

Treatment protocol

For the OFF condition (Samples 1-8), 1 mL of cells was diluted into 2 mL M9 containing 75 mM MgSO4 for a final concentration of 50 mM MgSO4. For the ON condition (Samples 11-18), 2 mL of cells were pelleted, washed twice with M9 + 10μM MgSO4, resuspended in M9 + 10 μM MgSO4, and then 1 mL of cells was diluted into 2 mL M9 + 10μM MgSO4. For trans-zeatin induction (Samples 9,10,19,20) cells were grown in 2 mL M9 + 1nM aTc and induced (Samples 19,20) by transferring 1mL cells to 2mL to identical media containing 1.5μM trans-zeatin (for final concentration of 1μM trans-zeatin). Uninduced samples (Samples 9,10) were generated by transferring 1mL cells to 2mL of just M9 + 1nM aTc. For all samples, RNA was harvested 30 minutes after cell induction.

Growth protocol

Cultures were grown overnight in M9 media containing 2 mM MgSO4 and then diluted 1:25 into fresh M9 containing 2 mM MgSO4 and grown for 2 hours to reach OD600 ~ 0.5.

Extracted molecule

total RNA

Extraction protocol

After 30 minutes of growth, cells from each condition were harvested by adding 1.8 mL culture to 200 μM cold stop solution (95% ethanol, 5% acid buffered phenol, 4 °C). The mixture was centrifuged for 30 s at 13,000 rpm on a benchtop centrifuge, and the supernatant was removed with the pellet flash frozen in liquid nitrogen and stored at -80 °C. To extract RNA, Trizol (Invitrogen) was heated to 65 °C, added directly to the pellet, and incubated at 65 °C for 10 minutes with shaking at 2000 rpm (Eppendorf Thermomixer). The mixture was frozen at -80 °C for at least 10 minutes. After thawing, cells were centrifuged at 15,000 rpm, 4 °C for 5 minutes, and the supernatant was removed into 400 μL ethanol. The mixture was applied to a DirectZol spin column (Zymo) and centrifuged for 30 s at 13,000 rpm. The columns were washed with DirectZol RNA prewash buffer twice (400 μL) and RNA wash buffer (700 μL) once before eluting in 90 μL DEPC water. 10 μL 10x Turbo DNAse buffer and 2 μL Turbo DNAse (Invitrogen) were added to the eluant. The mixture was digested at 37 °C for 20 minutes, followed by the addition of 2 μL more DNAse and another 20 minute incubation. Total volume was brought to 200 mL with DEPC water and combined with 200 μL acid-phenol:chloroform (IAA, Invitrogen), vortexed and centrifuged for 10 minutes at 21,000 g and 4 °C. The top (aqueous) layer was extracted and ethanol precipitated in 20 μL NaOAc (3M), 2 μL GlycoBlue (Invitrogen) and 600 μL cold ethanol. Precipitation mix was incubated at -80 °C for more than 4 hours before centrifuging for 30 minutes at 21,000 g and 4 °C. The pellet was washed twice with 500 μL cold 70% ethanol, then air dried and resuspended in 50 μL DEPC water. RNA integrity was validated on a 6% TBE-urea acrylamide Novex gel (Invitrogen) and yield was quantified by NanoDrop spectrophotometer. rRNA was removed with the RiboZero rRNA Removal Kit for Bacteria (Illumina).
RNA was fragmented and cDNA libraries were prepared using the KAPA RNA HyperPrep Kit (Roche) and sequenced on an Illumina HiSeq.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

sample 17

Data processing

Reads were mapped to the Echerichia coli MG1655 genome and plasmids with bowtie (default parameters)
RPKM ( Reads Per Kilobase Million) was calculated with custom python scripts
Supplementary_files_format_and_content: csv file containing RPKM for each gene in each sample. First line contains column titles

Submission date

Mar 20, 2019

Last update date

Sep 20, 2019

Contact name

Conor James McClune

E-mail(s)

conor.mcclune@gmail.com

Organization name

Massachusetts Institute of Technology

Department

Biology