|
| Status |
Public on Mar 05, 2022 |
| Title |
DglnA2 |
| Sample type |
RNA |
| |
|
| Source name |
Bacteria_LB grown
|
| Organism |
Escherichia coli |
| Characteristics |
strain: deltaglnA
|
| Treatment protocol |
The cells were grown in the presence or absence of 1 mM of manganese for 2.5 hours at 37°C
|
| Growth protocol |
Overnight cultures of E. coli strains were inoculated in the fresh LB medium at 1:100 dilution and grown initially for 1 hour to get the O.D. about 0.3 and then grown again for 2.5 hours in the presence or absence of 1 mM manganese at 37°C. 2.5 ml stop solution (5% phenol in ethanol) was added in 25 ml of growing culture for 10 minutes to prevent the RNA degradation and the cell pellets were collected by centrifuging at 5000 rpm for 10 minutes at 4°C. The pellets were washed with normal saline (0.9%) and stored by dissolving in 600ml RLT buffer before further processing.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA Extraction was perforemd using Qiagen RNeasy Mini kit (Cat. No. 74106) as per the manufacturer's recommendations.
|
| Label |
Cy3
|
| Label protocol |
The samples for Gene expression were labeled using Agilent Quick-Amp labeling Kit (p/n5190-0442). 500ng each of total RNA were reverse transcribed at 40°C using oligodT primer with T7 polymerase promoter converted to double stranded cDNA. Synthesized double stranded cDNA were used as template for cRNA generation. cRNA was generated by in vitro transcription and the dye Cy3 CTP(Agilent) was incorporated during this step. The cDNA synthesis and in vitro transcription steps were carried out at 40°C. Labeled cRNA was cleaned up using Qiagen RNesay columns (Qiagen, Cat No: 74106) and quality assessed for yields and specific activity using the Nanodrop ND-1000.
|
| |
|
| Hybridization protocol |
2000ng ng of labeled cRNA sample were fragmented at 60° C and hybridized on to a Agilent custom Microarray Gene expression 8x15k Array (AMADID: 068043). Fragmentation of labeled cRNA and hybridization were done using the Gene Expression Hybridization kit of (Agilent Technologies, In situ Hybridization kit, Part Number 5190-0404). Hybridization was carried out in Agilent’s Surehyb Chambers at 65° C for 16 hours. The hybridized slides were washed using Agilent Gene Expression wash buffers (Agilent Technologies, Part Number 5188-5327)
|
| Scan protocol |
Agilent Microarray Scanner (Agilent Technologies, Part Number G2600D).
|
| Description |
Gene expression_DglnA strain_minus manganase_2
|
| Data processing |
Images were quantified using Feature Extraction Software ( Agilent). Feature extracted raw data was analyzed using in-house coded R Scripts (https://cran.r-project.org/). Normalization of the data was done in R-Scripts .
|
| |
|
| Submission date |
Oct 27, 2021 |
| Last update date |
Mar 06, 2022 |
| Contact name |
Dipak Dutta |
| E-mail(s) |
dutta@imtech.res.in, ddutta25@gmail.com
|
| Phone |
01726665263
|
| Organization name |
CSIR IMTECH
|
| Department |
Molecular Biochemistry
|
| Lab |
Transcription and replication biology
|
| Street address |
Sector 39-A
|
| City |
Chandigarh |
| State/province |
Chandigarh |
| ZIP/Postal code |
160036 |
| Country |
India |
| |
|
| Platform ID |
GPL30904 |
| Series (1) |
| GSE186657 |
The global transcriptomic profile in the manganese-stressed wild type, DglnA and DmntPDglnA E. coli strains |
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