|
| Status |
Public on Mar 22, 2016 |
| Title |
exp_transcriptome |
| Sample type |
RNA |
| |
|
| Source name |
bacterial cells
|
| Organism |
Escherichia coli str. K-12 substr. MG1655 |
| Characteristics |
genotype: Wild-type
growth phase: Mid-Exponential
|
| Treatment protocol |
Cells from 12.5 ml culture were harvested by centrifugation
|
| Growth protocol |
Cells were grown in LB medium at 37 C with aeration, until mid-exponential phase (OD600=0.95) or stationary phase (OD600=2.4).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was purified using the RNeasy ® Mini Kit (Qiagen ® 74104) following the manufacturer’s recommendations. Isolated RNAs (10 ug) were used for Random Primer cDNA synthesis using SuperScript IITM Reverse Transcriptase, (Invitrogen Life Technologies 18064-071). RNA was removed by treating the reaction mixture with 1N NaOH followed by neutralizing with 1N HCl. cDNAs were purified using MiniElute ® PCR Purification columns (Qiagen ® 28004) and fragmented to 50-200 bp by 0.6U/ug of DNase I for 10 minutes at 37°C in 1X One-Phor-All buffer (Amersham Biosciences 27-0901-02). DNase I was inactivated by heating at 98°C for 10 minutes.
|
| Label |
biotin
|
| Label protocol |
Fragmented cDNAs were then 3’ termini biotin labeled using the GeneChip ® DNA Labeling Reagent (Affymetrix 900542) and 60U of Terminal Deoxynucleotidyl Transferase (Promega M1875) at 37°C for 60 minutes. The labeling reaction was stopped by the addition of 0.5M EDTA. Labeled cDNA fragments (3ug) were then hybridized for 16 hours (60 rpms) at 45°C to tiling array chips (Ecoli_Tab520346F, GPL8585).
|
| |
|
| Hybridization protocol |
According to Affymetrix specifications
|
| Scan protocol |
Scanned using a Genechip Scanner 3000 (Affymetrix) according to Affymetrix specifications
|
| Data processing |
Probe intensities were corrected for background by subtracting the median intensities of control (non-E. coli) probes having the same G+C content.
.txt files with 3 columns. Column 1: probe sequence. Column 2: start position of probe on E. coli chromosome. Column 3 = log2 background-corrected intensity values for each probe
|
| |
|
| Submission date |
Feb 08, 2016 |
| Last update date |
Mar 23, 2016 |
| Contact name |
Avantika Lal |
| E-mail(s) |
avantika@ncbs.res.in
|
| Organization name |
National Centre for Biological Sciences
|
| Street address |
GKVK, Bellary Road
|
| City |
Bangalore |
| ZIP/Postal code |
560065 |
| Country |
India |
| |
|
| Platform ID |
GPL8585 |
| Series (1) |
| GSE77686 |
E. coli transcriptome in stationary and mid-exponential phase |
|
