|
| Status |
Public on Aug 31, 2018 |
| Title |
Replicate 1: LIX48 versus LIX47 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
H37Ra19 control_R2
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
strain/genotype: empty vector control strain
|
| Treatment protocol |
integrative plasmid pJFR19-devR (Hyg resistant) was electroporated in H37Ra and H37Rv to yield LIX48 and LIX50 strain respectively. Empty vector pJFR19 was electroporated into H37Ra and H37Rv to yield the respective control strains named here LIX47 and LIX49 respectively
|
| Growth protocol |
The recombinant H37Ra and H37Rv strains were cultivated in Middlebrook 7H9 medium supplemented with 0.05% Tween 80 and 10% ADS (0.5% albumin, 0.2% dextrose, 0.085% Saline) (7H9T medium) or on Middlebrook 7H10 agar plates at 37ºC to an OD600 ~0.2-0.3
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRI reagent (Ambion) following the manufacturers instructions.
|
| Label |
cy3
|
| Label protocol |
Poly (A)-tails were added to the 3’-end of RNA by using A-plus Poly (A) polymerase tailing kit (Epicentre Biotechnologies). Then the samples were labeled using Agilent Quick-Amp labeling Kit (p/n:5190-0444). Five hundred nanograms each of the samples were incubated with reverse transcription mix at 42°C and converted to double stranded cDNA primed by oligodT with a T7 polymerase promoter. The cleaned up double stranded cDNA were used as template for cRNA generation. cRNA was generated by in vitro transcription and the dye Cy3-CTP(Agilent) and Cy5-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 and quality assessed for yields and specific activity.
|
| |
|
| Channel 2 |
| Source name |
H37Ra85_DevR_R2
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
strain/genotype: H37Ra devR overexpressing strain
|
| Treatment protocol |
integrative plasmid pJFR19-devR (Hyg resistant) was electroporated in H37Ra and H37Rv to yield LIX48 and LIX50 strain respectively. Empty vector pJFR19 was electroporated into H37Ra and H37Rv to yield the respective control strains named here LIX47 and LIX49 respectively
|
| Growth protocol |
The recombinant H37Ra and H37Rv strains were cultivated in Middlebrook 7H9 medium supplemented with 0.05% Tween 80 and 10% ADS (0.5% albumin, 0.2% dextrose, 0.085% Saline) (7H9T medium) or on Middlebrook 7H10 agar plates at 37ºC to an OD600 ~0.2-0.3
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using TRI reagent (Ambion) following the manufacturers instructions.
|
| Label |
cy5
|
| Label protocol |
Poly (A)-tails were added to the 3’-end of RNA by using A-plus Poly (A) polymerase tailing kit (Epicentre Biotechnologies). Then the samples were labeled using Agilent Quick-Amp labeling Kit (p/n:5190-0444). Five hundred nanograms each of the samples were incubated with reverse transcription mix at 42°C and converted to double stranded cDNA primed by oligodT with a T7 polymerase promoter. The cleaned up double stranded cDNA were used as template for cRNA generation. cRNA was generated by in vitro transcription and the dye Cy3-CTP(Agilent) and Cy5-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 and quality assessed for yields and specific activity.
|
| |
|
| |
| Hybridization protocol |
300ng of Cy3 labeled sample and 300ng of Cy5 labeled sample were fragmented and hybridized. Fragmentation of labeled cRNA and hybridization were done using the Gene Expression Hybridization kit of Agilent (Part Number 5188-5242). 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 (Part No: 5188-5327)
|
| Scan protocol |
scanned using the Agilent Microarray Scanner G2505C at 5 micron resolution. Data extraction from Images was done using Agilent Feature Extraction software v 10.5.1.1
|
| Data processing |
Data Analysis was performed using Genespring GX Software using LOWESS normalization.(Gene based analysis was performed).
|
| |
|
| Submission date |
Sep 06, 2017 |
| Last update date |
Aug 31, 2018 |
| Contact name |
Genotypic technology |
| E-mail(s) |
sudha.rao@genotypic.co.in
|
| Organization name |
Genotypic Technology
|
| Street address |
259, Apoorva 4th cross,80 feet Road,RMV 2ND STAGE
|
| City |
Bangalore |
| State/province |
Karnataka |
| ZIP/Postal code |
560094 |
| Country |
India |
| |
|
| Platform ID |
GPL23988 |
| Series (1) |
| GSE103551 |
LIX48 (H37Ra-devR overexpression) versus LIX47 (control strain) and LIX50 (H37Rv-devR overexpression) versus LIX49 (control strain) |
|
