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Sample GSM5856987

Query DataSets for GSM5856987

Status

Public on Dec 03, 2022

Title

PknH_IND3

Sample type

SRA

Source name

H37Rv Mycobacterium tuberculosis

Organism

Mycobacterium tuberculosis

Characteristics

growth phase: Stationary
genotype/variation: PknH_IND

Treatment protocol

Bacteria were harvested at early stationary phase.

Growth protocol

Mycobacteria tuberculosis strain H37Rv (ATCC 27294) (WT, STPK knockout, and STPK induction) were grown on Middlebrook 7H9 medium (Difco) with 0.2 % glycerol and 10% oADC supplement.

Extracted molecule

total RNA

Extraction protocol

RNA was isolated using published protocols (Rustad TR, Harrell MI, Liao R, Sherman DR (2008) The Enduring Hypoxic Response of Mycobacterium tuberculosis. PLoS ONE 3(1): e1502). In summary, frozen cell pellets Pellets were resuspended in Trizol, transferred to a tube containing Lysing Matrix B (QBiogene, Inc.), and vigorously shaken at max speed for 30 sec in a FastPrep 120 homogenizer (Qbiogene) three times, with cooling on ice between steps. This mixture was centrifuged at max speed for 1 min and the supernatant was transferred to a tube containing 300 mL chloroform and Heavy Phase Lock Gel (Eppendorf North America, Inc.), inverted for one minute, and centrifuged at max speed. The aqueous phase was then precipitated with 270 mL isopropanol and 270 mL high salt solution (0.8M Na citrate, 1.2M NaCl). RNA was cleaned using an RNeasy kit following manufacturer’s recommendations (Qiagen). Messenger RNA was enriched using the RiboZero rRNA removal (bacteria) magnetic kit (Illumina Inc.) according to manufacturer's instructions.
Libraries were prepared for Illumina sequencing using the NEBNext Ultra RNA Library Prep Kit for Illumina (New England Biolabs) according to manufacturer's instructions and using the AMPure XP reagent (Agencourt Bioscience Corporation) for size selection and cleanup of adaptor-ligated DNA. We used the NEBNext Multiplex Oligos for Illumina (Dual Index Primers Set 1) to barcode the libraries associated with each replicate and multiplexing up to 48 libraries per sequencing run. The prepared libraries were quantified using the Kapa qPCR quantification kit, and were sequenced at the University of Washington Northwest Genomics Center with the Illumina NextSeq 500 High Output v2 Kit (Illumina Inc).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Raw FASTQ read data processing was carried out using a custom processing pipeline that harnesses the Bowtie 2 utilities, which is available at https://github.com/sturkarslan/DuffyNGS, and https://github.com/sturkarslan/DuffyTools, as previously described (PMID: 25784701)
The pipeline consists of 2 stages: 1) a pre-alignment filtering step to remove unwanted transcripts such as rRNA, and 2) a genomic alignment step using Bowtie2 with the command line option 'very-sensitive.'
BAM files from step 2 are combined into read depth wiggle tracks that record both uniquely mapped and multiply mapped reads from each of the forward and reverse strangs of the genome. Multiply mapped reads are prorated over all highest-quality aligned locations. Transcript abundance is quantified by summing total reads landing inside annotated gene boundaries, expressed by RPKM.
Genome_build: MT_H37Rv_V3
Supplementary_files_format_and_content: Transcript abundance is quantified by summing total reads landing inside annotated gene boundaries, expressed by RPKM.

Submission date

Feb 02, 2022

Last update date

Dec 03, 2022

Contact name

Christoph Grundner

E-mail(s)

christoph.grundner@seattlechildrens.org

Phone

2099155648

Organization name

Seattle Children's Research Institute