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| Status |
Public on Oct 15, 2024 |
| Title |
F50 fungus in Phosphate buffer (b) |
| Sample type |
SRA |
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| Source name |
Fungus
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| Organism |
Penicillium sp. F50 |
| Characteristics |
cell type: Fungus
treatment: F50-Mtb Control
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| Treatment protocol |
For co-cultures, 450 µL of 1X (for F2/F50/F51 fungus), 0.1X (for C7 fungus) and 0.001X (for F31 fungus) strength of Mtb freezer stock resuspended in phosphate buffer (pH 6.0) were added to fungal cultures on the next day and the cultures were incubated at 32°C at 45 rpm
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| Growth protocol |
Fungal Mono-/ and Co-culture: Fungal biomass from axenic and co-cultures of the induced potential hits was obtained by inoculating about 300,000 spores in 4 mL Potato Dextrose Broth (PDB) media in 6 well plates (Corning Inc.) for F2/F2+Mtb; C7/C7+Mtb; F31/F31+Mtb; F50/F50+Mtb; F51/F51+Mtb. The cultures were grown at 28°C for one day. For co-cultures, 450 µL of 1X (for F2/F50/F51 fungus), 0.1X (for C7 fungus) and 0.001X (for F31 fungus) strength of Mtb freezer stock resuspended in phosphate buffer (pH 6.0) were added to fungal cultures on the next day and the cultures were incubated at 32°C at 45 rpm. All culture sets were carried out in at least two biological replicates. In contrast, fungal cultures with only phosphate buffer (pH 6.0) added were used as controls. Saturated Mtb freezer stock preparation for Co-cultures: 2 L detergent and BSA-free 7H9 media (supplemented with 4 g/L Glucose, 0.81 g/L NaCl and 0.3 g/L Casitone) was inoculated with 200 mL of wild-type Mtb H37Rv (OD600 1.0) in roller bottles and incubated at 37°C for 2 weeks. The cells were harvested by centrifuging at 4000 rpm for 30 min and were washed with 200 mL phosphate buffer saline (PBS). The cells were re-washed as above and finally resuspended in 200 mL PBS to form 10X concentrate. The cells were homogenized using glass beads (4 mm, 20 mL) by vortexing for 5 min. The 10X cell suspension was then aliquoted into cryovials and frozen at -80°C for further use. When used at different concentrations for co-culturing with fungus, the concentrated Mtb stocks were serially diluted in phosphate buffer (pH 6.0) and homogenized by using glass beads (4mm, 5 mL) for preparing a 10 mL suspension in 50 mL falcon tube followed by vortexing for at least 2 min.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Mycelia was harvested on days 6, 9 and 12 by centrifuging at 4000 rpm for 20 min followed by a brief washing with DEPC-treated water (10 mL, 4000 rpm, 20 min). The media from both mono- and co-cultures was filtered through 0.2 µm microplate filter (Millipore) from each set and validated for the growth inhibition activity against pOLYG-mScarlet Mtb H37Rv on respective days. The harvested fungal biomass was ground to fine powder in liquid nitrogen with a mortar-pestle, which was then transferred to 2-3 mL TRIzol Reagent (Invitrogen) to form a slurry and finally thawed. The samples were pipetted several times to ensure complete homogenization and were split into 2-3 RNase-free microcentrifuge tubes (1 mL each) and incubated for 5 min at RT. Chloroform (300 µL) was added to each tube and these were shaken vigorously. The samples were centrifuged at 13,000 x g at 4°C for 15 min. To the upper aqueous layer, an equal volume of 70% ethanol was added to precipitate the RNA, vortexed and the solution was loaded onto spin cartridge columns provided by PureLink RNA isolation kit (Invitrogen). Further purification steps were done according to the manufacturer’s guidelines with an on-column DNase I treatment (Zymo Research). The RNA was finally eluted in 40 μL DEPC-treated RNase-free water (Ambion, Invitrogen) and stored at -80°C. RNA concentration and purity were analyzed using Nanodrop spectrophotometer (Thermo Scientific) followed by visual assessments using Bioanalyzer (Agilent 2100) and denaturing formaldehyde agarose gel electrophoresis (1.3% agarose, formaldehyde in 1X MOPS buffer). Total RNA (at least in two biological replicates) from mono- and co-culture conditions were used for mRNA sequencing following ribosomal RNA depletion and cDNA Library prep
Libraries for mRNA sequencing were constructed at ZymoResearch from total RNA sample using the Zymo-Seq RiboFree Total RNA Library Prep Kit (Cat # R3000) according to the manufacturer’s instructions. Briefly, RNA was reverse transcribed into cDNA, which was followed by ribosomal RNA depletion. After that a partial P7 adapter sequence was ligated at 3’ end of cDNAs, followed by second strand synthesis and partial P5 adapter ligation to 5’ end of the double stranded DNAs. Lastly, libraries were amplified to incorporate full length adapters under the following conditions: initial denaturation at 95°C for 10 min; 10 - 16 cycles of denaturation at 95°C for 30 sec, annealing at 60°C for 30 sec, and extension at 72°C for 60 sec; and final extension at 72°C for 7 min. Successful library construction was confirmed with Agilent’s D1000 ScreenTape Assay on TapeStation
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
F50Day6_minus_Tb_b
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| Data processing |
RNA-Seq libraries were sequenced on an Illumina NovaSeq 6000 to a sequencing depth of at least 30 million read pairs (150 bp paired-end sequencing) per sample.
The Zymo Research RNA-Seq pipeline was originally adapted from nf-core/rnaseq pipeline v1.4.2. The pipelines was built using Nextflow. Adapter and low-quality sequences were trimmed from raw reads using Trim Galore! v0.6.6. Trimmed reads were aligned to the reference genome using STAR v2.6.1d. BAM file filtering and indexing was carried out using SAMtools v1.9. RNAseq library quality control was implemented using RSeQC v4.0.0 and QualiMap v2.2.2-dev. Duplicate reads were marked using Picard tools v2.23.9. Reads overlapping with exons were assigned to genes using featureCounts v2.0.1.
Assembly: JAYXSA000000000
Supplementary files format and content: merged_gene_counts.txt: raw counts file
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| Submission date |
Jun 30, 2024 |
| Last update date |
Oct 15, 2024 |
| Contact name |
Tovah E Markowitz |
| Organization name |
NIAID
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| Department |
Integrated Data Sciences Section (IDSS)
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| Lab |
NIAID Collaborative Bioinformatics Resource (NCBR)
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| Street address |
10 Center Dr, Building 29B
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20814 |
| Country |
USA |
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| Platform ID |
GPL34662 |
| Series (1) |
| GSE271124 |
Co-cultivation induces elicitation of unique Fungus-derived Natural products that leads to thiol stress mediated killing of Mycobacterium tuberculosis [F50] |
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| Relations |
| BioSample |
SAMN42180430 |
| SRA |
SRX25159976 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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