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| Status |
Public on Aug 03, 2020 |
| Title |
M5A_1Wk_Exp1A_M5A_S9 |
| Sample type |
SRA |
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| Source name |
bacterial culture
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| Organism |
Mycobacterium tuberculosis variant bovis BCG |
| Characteristics |
strain: Pasteur ATCC 35734
growth stage: Intercellular aggregation
age: 7 days
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| Growth protocol |
Biofilms (which includes bacteria attached to the plastic wells and surface pellicles) for RNA extraction of BCG strains were cultured in Sauton media (containing the following g/L of anhydrous compounds from Sigma unless indicated otherwise): L-asparagine (4.0), citric acid (2.0), KH2PO4 (0.5), MgSO4 (0.5), ferric ammonium citrate (0.05), 1% ZnSO4 (1 mL), glycerol (60 mL), pH 7.0, and incubated at 37ºC, static, 5% CO2. All cultures were started at OD600nm 0.03. After 1, 7, and 10 days of incubation, two culture flasks were used to harvest the whole surface pellicle and biofilm attached to the wells (these samples are referred to as “biofilms”).
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| Extracted molecule |
total RNA |
| Extraction protocol |
For each 1 mg of wet mass, 1 ml Trizol (Invitrogen) was added, and lysis was performed using 0.4 ml of 0.1 mm zirconia/silica beads by three cycles at full speed in a bead beater, including incubation in ice for 30 s between cycles of beating, followed by 1 min of centrifugation at 13,000 rpm, 4°C. Trizol supernatant was transferred to a heavy-lock phase tube (Eppendorf) containing 200 μl chloroform (Sigma), and vigorously shaken for 15 s, during 2 min. After 10 min at room temperature (RT), we centrifuged at 13,000 rpm, RT, for 5 min, and the supernatant (volume ∼540 μl) was transferred to 1.5 ml tubes, to which 270 μl of 2-propanol (Sigma) and 270 μl of 3 M sodium acetate pH 5.2 (Molecular biology grade, Sigma) were added, followed by mixing and incubation overnight at 4°C. Samples were centrifuged at 13,000 rpm, 4°C, 10 min, and the nucleic acids pellet was washed twice with 70% ethanol. After drying and dissolving in RNAse-free water (Sigma), we quantitated nucleic acids using a UV spectrophotometer. We performed genomic DNA digestion in solution using RQ1 DNase (Promega) following the manufacturer’s recommendation, and further clean-up was performed using RNeasy kit (Qiagen) according to their recommended protocol. RNA was eluted and quantitated
RNA was used to prepare cDNA using Nugen’s Ovation RNA-Seq System via single primer isothermal amplification (Catalogue # 7102-A01) and automated on the (BRAVO NGS liquid handler from Agilent). cDNA was quantified on the Nanodrop (Thermo Fisher Scientific). Using Kapa Biosystem’s DNA Hyper Plus library preparation kit, (KK8514) cDNA was enzymatically sheared to approximately 150 bp fragments, end repaired and A-tailed. Illumina-compatible adapters with unique indexes (IDT #00989130v2) were ligated on each sample individually. The adapter ligated molecules were cleaned using Kapa pure beads (Kapa Biosciences, KK8002), and amplified with Kapa’s HIFI enzyme (KK2502). Each library was then analyzed for fragment size on an Agilent’s Tapestation, and quantified by qPCR (KAPA Library Quantification Kit, KK4835) on Thermo Fisher Scientific’s Quantstudio 5 before multiplex pooling and sequencing a 2x75 flow cell on the NextSeq500 platform (Illumina) at the ASU’s Genomics Core facility.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Samples taken at 7 days of culture
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| Data processing |
Raw FASTQ read data were processed using the R package DuffyNGS.
Genome_build: ASM944v1
Supplementary_files_format_and_content: tab-delimited text file includes RPKM for each sample
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| Submission date |
May 07, 2020 |
| Last update date |
Aug 03, 2020 |
| Contact name |
Eliza Peterson |
| Organization name |
Institute for Systems Biology
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| Lab |
Baliga
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| Street address |
401 Terry Ave N
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| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
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| Platform ID |
GPL28494 |
| Series (1) |
| GSE150030 |
Transcriptional portrait of M. bovis BCG during in vitro biofilm production shows genes differentially expressed during intercellular aggregation and substrate attachment. |
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| Relations |
| BioSample |
SAMN14848243 |
| SRA |
SRX8287153 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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