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| Status |
Public on Jan 15, 2021 |
| Title |
TV_M7_Cel_Lignin (TV_21) |
| Sample type |
SRA |
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| Source name |
Trametes versicolor
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| Organism |
Trametes versicolor |
| Characteristics |
treatment: 5 g/L cellobiose, 25 mg lignin/25 mL
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| Treatment protocol |
Media preparation:
The media utilized to maintain the strains and initiate seed cultures was adapted YMPG-agar medium. Adapted YMPG medium consisted of 2 g yeast extract, 10 g malt extract, 2 g peptone, 10 g glucose, 2 g KH2PO4, and 1 g MgSO4 x 7H2O per liter in H2O on 1.5 % agar plates. This medium was autoclaved for 30 min at 121°C and plated. The liquid pre-seed and seed cultures consisted of the same media but excluding agar.
For the experimental campaigns, we developed a minimal and defined medium which is an adapted version of Czapek-Dox medium (CDM). CDM consisted of 2 g ammonium tartrate, 0.5 g MgSO4, 1 g KH2PO4, 0.5 g KCl, 1 mL sterile vitamins supplement (ATCC® MD-VS™), and 1 mL trace elements solution per liter in H2O. The trace elements solution contains 0.1 g Na2B4O7 x 10 H2O, 0.07 g ZnSO4 x 7 H2O, 0.01 g CuSO4 x 5 H2O, 0.01 g MnSO4 x 7 H2O, 0.05 g FeSO4 x 7 H2O, and 0.01 (NH4)6MoO2 x 4 H2O per liter in H2O. Trace elements were filter-sterilized through 0.2 µM PES filters (Millipore) and further added to CDM after autoclaving (30 min at 121°C).
Carbon sources and concentrations added to CDM for submerged-state cultivations were specific to each experimental campaign. Specifically, concentrated stocks of cellobiose (100 g/L), aromatic compounds such as 4-HBA (10 g/L), vanillic acid (10 g/L), and syringic acid (10 g/L) were prepared by dissolving the compound in H2O and slowly adjusting the pH to 7 with 4 M NaOH. These compounds were subsequently filter sterilized using 0.22 µm PES Steriflip filter units (Millipore) and added to CDM. Lignin isolated from poplar was resuspended in H2O (in aliquots of 25 mg in 0.5 mL of H2O), UV-sterilized, and added to CDM. When poplar was the substrate, 2 g of milled poplar were autoclaved for 30 min at 121°C in individual flasks containing 8 mL of H2O. Then, CDM without cellobiose was added to the poplar mixture.
Cultivations for multi-omic experiments and sample preparation:
G. subvermispora and T. versicolor cultivations were conducted in 250 mL flasks containing 25 mL of CDM with cellobiose (5 g/L) and incubated at 28°C in static conditions using a humidity-controlled incubator (˃50 % humidity). When the remaining cellobiose was ~50-80% of the initial concentration (12 d and 5 d for G. subvermispora and T. versicolor, respectively), we induced the cultivations with different substrates, 4-HBA (2 mM), syringic acid (2 mM), or lignin (25 mg, see media preparation in Section 2). Poplar was also used as an inducing substrate. However, poplar (2 g) was present since the beginning of the cultivation in CDM without cellobiose (see media preparation in Section 2). Antioxidants (5 mM ascorbic acid and 1 mM α-tocopherol) were added in the media 30 min before the induction, including the media that contained poplar. Cultivations were harvested 48 h after the induction, corresponding to 14 d and 7 d for G. subvermispora and T. versicolor, respectively. Each experimental condition, including controls, was performed in triplicate. In this case the controls were (1) cultivations conducted in CDM and cellobiose as the sole carbon source and (2) non-inoculated media.
Transcriptomic samples were generated by centrifugation at 6,000 x g for 10 min, followed by flash freezing the pellets in liquid nitrogen after discarding the supernatants. Samples were stored at -80°C.
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| Growth protocol |
Fungal seed preparation:
To start each experiment with fungal suspensions in an equivalent metabolic state, seed cultures were prepared as follows. An agar square (~0.5 cm2) with fungal mycelia was taken from agar plates maintained at 4°C, deposited on a fresh YMPG-agar plate (mycelia in contact with the agar), and incubated for 7 days at 28°C. Then, 4-5 agar squares (~0.5 cm2) with mycelia from the fungal mat edges were inoculated in 50 mL liquid culture of YMPG (pre-seed cultures) in non-baffled 250 mL flasks and incubated at 220 rpm and 28°C for 7 days. Resulting fungal pellets were homogenized (10 s, speed 6) using an Omni Mixer 115V with a 50 mL SS Chamber PTFE, and 2 mL of the resulting fungal suspension were re-inoculated and cultured in YMPG under the same conditions for 5 days (seed cultures). Fungal pellets were homogenized as described previously and a ratio of 1:12.5 (v:v) of fungal suspension per media volume was used to inoculate the corresponding cultivations. Glucose content was analyzed in the seed cultures before inoculation in a rapid biochemistry analyzer (2900D YSI) to verify that the cultures had not reached glucose starvation.
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| Extracted molecule |
total RNA |
| Extraction protocol |
The total RNA was extracted using Qiagen RNeasy Kits (cat#74104) and RNA samples were assessed using the Agilent 2100 Bioanalyzer. The RNA with RIN > 7 was chosen to proceed with ribosomal rRNA remover using Ribo-zero yeast (illumina cat#MRZY1306).
cDNA libraries for illumina NextSeq550 platform were generated using TruSeq Stranded Total RNA (cat# 20020596) according to the manufacturer's protocol.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Data processing |
Read quality was assessed using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/)
Read-trimming was conducted using bbduk (http://jgi.doe.gov/data-and-tools/bb-tools/) with parameters trimq=10, qtrim="rl", maq=10.
Reads were aligned to the Trametes versicolor and Gelatoporia subvermispora genomes using bowtie2 (http://bowtie-bio.sourceforge.net/bowtie2/index.shtml). using parameters -local, --sensitive-local
Aligned reads were mapped to genes using htseq-count (https://pubmed.ncbi.nlm.nih.gov/25260700/) with parameters -a=1, --mode="union".
Genome_build: Trametes versicolor genome https://www.ncbi.nlm.nih.gov/assembly/GCF_000271585.1/
Genome_build: Gelatoporia subvermispora genome https://www.ncbi.nlm.nih.gov/assembly/GCA_000320605.2/
Supplementary_files_format_and_content: read counts per gene
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| Submission date |
Jun 02, 2020 |
| Last update date |
Jan 16, 2021 |
| Contact name |
Davinia Salvachua |
| Organization name |
National Renewable Energy Laboratory
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| Street address |
15013 Denver West Parkway
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| City |
Golden |
| State/province |
CO |
| ZIP/Postal code |
80401 |
| Country |
USA |
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| Platform ID |
GPL28618 |
| Series (1) |
| GSE151629 |
Intracellular pathways for lignin catabolism in white-rot fungi |
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| Relations |
| BioSample |
SAMN15081406 |
| SRA |
SRX8453281 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4586820_TV_21.txt.gz |
109.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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