|
| Status |
Public on Sep 07, 2021 |
| Title |
Mtb (phosphate limited) rep2 |
| Sample type |
SRA |
| |
|
| Source name |
Mtb phosphate limited
|
| Organism |
Mycobacterium tuberculosis |
| Characteristics |
condition: phosphate limited
|
| Treatment protocol |
Log phase cells were harvested and resuspended in either phosphate enriched or phosphate limited medium and grown for 72 hours under shaking at 37◦C.
|
| Growth protocol |
Mtb was grown to log phase in Middle Brook 7H9 medium containing 10% ADC.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted by homogenising in a Mini bead beater and purified with the Qiagen RNeasy kit.
~2.0 µg of total RNA was taken for rRNA depletion using Ribo-Zero rRNA Removal Kit (Bacteria). ~30 ng of Qubit quantified ribodepleted RNA was taken for fragmentation and priming. The fragmented and primed mRNA was further subjected to first strand synthesis in the presence of Actinomycin D (Gibco, Life Technologies, CA, USA) followed by second strand synthesis. The double stranded cDNA was purified using JetSeq magnetic beads (Bio, # 68031). The purified cDNA was end-repaired, adenylated and ligated to Illumina multiplex barcode adapters as per NEBNext® Ultra™ Directional RNA Library Prep Kit protocol. The adapter ligated cDNA were purified JetSeq beads and were subjected to 15cycles of Indexing-PCR (37 ˚C for 15mins followed by denaturation at 98˚C for 30 sec, cycling (98˚C for 10sec, 65˚C for 75sec) and 65˚C for 5mins) to enrich the adapter-ligated fragments. The final PCR products (sequencing library) were purified with JetSeq beads, followed by libraries quality control check. The Illumina-compatible sequencing libraries were initially quantified by Qubit fluorometer (Thermo Fisher Scientific, MA, USA) and its fragment size distribution were analyzed on Agilent TapeStation.
Transcriptomic profiling was done using Illumina-compatible NEBNext® Ultra™ Directional RNA Library Prep Kit ((NEB, #E7420S) ) at Genotypic Technology Pvt. Ltd., Bangalore, India.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
HiSeq X Ten |
| |
|
| Description |
1B
|
| Data processing |
Libraries were sequenced at Genotypic Technology Pvt. Ltd., on an Illumina Hiseq XTen platform by a 150bp paired-end reads.
DESeq software was used to carry out the transcriptomic analysis.
Genome_build: Mycobacterium tuberculosis H37Rv genome, accession: NC_000962
Supplementary_files_format_and_content: GT_SO_7951_Expression_Matrix.xlsx: Expression values in RC_matrix sheet indicate raw read count of genes generated by HTSeq; in the RPM_matrix sheet, reads are normalized to the total reads (i.e. reads per million) for each sample.
|
| |
|
| Submission date |
Sep 05, 2021 |
| Last update date |
Sep 22, 2021 |
| Contact name |
Manikuntala Kundu |
| E-mail(s) |
manikuntala.kundu@gmail.com
|
| Organization name |
Bose Institute
|
| Street address |
93/1, Acharya Prafulla Chandra Road
|
| City |
Kolkata |
| State/province |
West Bengal |
| ZIP/Postal code |
700009 |
| Country |
India |
| |
|
| Platform ID |
GPL27661 |
| Series (1) |
| GSE183469 |
Global transcriptomic profiling of Mycobacterium tuberculosis (Mtb) H37Rv in phosphate limited or phosphate enriched media |
|
| Relations |
| BioSample |
SAMN21238181 |
| SRA |
SRX12020282 |
