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| Status |
Public on Mar 13, 2022 |
| Title |
Fe/Cysteine rep D |
| Sample type |
SRA |
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| Source name |
whole cells
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| Organism |
Methanosarcina barkeri |
| Characteristics |
strain: MS
fe source: FeCl2
fe source: cysteine
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| Treatment protocol |
Cultures of M. barkeri MS were grown in quadruplicate on three different sources of Fe and S for RNA-Seq analysis: 2 mM FeS2, 20 µM FeCl2 and 2 mM L-cysteine, and 20 µM FeCl2 and 2 mM Na2S. Cells were harvested at mid-log growth phase, as determined by CH4 production, and final biomass was estimated by DNA quantification.
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| Growth protocol |
M. barkeri strain MS was obtained from the American Type Culture Collection (ATCC 43569). Growth medium was prepared without added Fe and S in MilliQ water and using acid (10% HNO3) washed glassware. M. barkeri MS was grown in low-salinity medium (in g L-1: NaCl, 1.00; MgCl2 · 6H2O, 0.40; NH4Cl, 0.54; KCl, 0.50; CaCl2 · 2H2O, 0.10). The base salts solution was boiled for 10 minutes then purged with N2 gas passed over a heated (200 °C) and H2-reduced column containing reduced copper shavings for 1 hr L-1. After sparging, base salts medium was moved to an anaerobic chamber and allowed to cool to room temperature (~20 °C). Once cooled, NaHCO3 (2.00 g L-1) was added and the pH was adjusted to 7.0 with anoxic 2N HCl. Next, 75 mL of the base salts medium were dispensed into 165 mL serum bottles, sealed with black butyl stoppers, and capped with aluminum crimp caps. Sealed serum bottles were removed from the anaerobic chamber, the headspace was exchanged for 15 minutes with N2:CO2 (80%:20%) gas passed over a heated copper column, and then autoclaved. After autoclaving, a 100X sterile and anoxic phosphate solution containing 0.35 g L-1 K2HPO4 and 0.23 g L-1 KH2PO4 was added to each bottle of base salts medium. Prior to inoculation, the base salts medium used to cultivate both methanogen strains was amended with 1% (v/v) Wolfe’s vitamins and 1% SL-10 trace metals (no added Fe containing components). Cultures of both M. barkeri strains were provided with 0.5% (v/v) methanol and 40 mM acetate as a methanogenesis substrate and carbon source, respectively, and grown anaerobically with a N2:CO2 (80%:20%) headspace pressurized to 1.72 bar.
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| Extracted molecule |
total RNA |
| Extraction protocol |
To harvest biomass, M. barkeri MS cells were subjected to vacuum filtration at low pressure (5 psi) onto a 47 mm 0.2-µm Supor 200 PES filter (Pall, Port Washington, NY) within an anaerobic chamber. Using flame-sterilized scissors, each filter was cut in half and each half transferred to a 1.5 mL cryotube before being removed from the anaerobic chamber and immediately flash frozen in liquid nitrogen. Frozen cells were then stored at -80 °C until processing. Total RNA from M. barkeri was extracted using TRIzol reagent (Invitrogen) following the manufacturer’s protocol with slight modification. Individual frozen half filters containing M. barkeri cells were transferred to a Lysis E tube (MP Biomedicals) on ice and 1 mL of TRIzol was immediately added to the frozen filter. TRIzol-treated M. barkeri cells were subjected to three cycles of 40 seconds of bead beating followed by resting for five minutes at room temperature (~20 °C). Two hundred µL of molecular grade chloroform was added to each tube and each tube was inverted to mix, incubated at room temperature for three minutes, and centrifuged for 15 minutes at 12,000 x g at 4 °C. The upper aqueous phase containing RNA was carefully transferred to a clean 2 mL tube. To precipitate RNA, 0.5 mL of pre-chilled (4 °C) 100% molecular grade isopropanol was added to each tube and tubes and their contents were incubated on ice for 10 minutes followed by centrifugation for 10 minutes at 12,000 x g at 4 °C. The supernatant was discarded, and 1 mL of 75% molecular grade ethanol was added to wash the RNA. After centrifugation for 5 minutes at 7,500 x g at 4 °C, the supernatant was removed, and the RNA pellet was air dried for 5-10 minutes. Total RNA was dissolved in 50 µL RNA-grade water (Fisher Scientific, Waltham, MA) by incubating in a 55 °C heat block for 10 minutes. RNA was treated with Turbo DNase (Invitrogen) to remove residual DNA per manufacturer’s instructions. After DNase treatment, the remaining total RNA was subjected to a second round of isopropanol precipitation, ethanol wash, and resuspension as described above.
Quality control was performed with a Bioanalyzer (Agilent, CA, USA), and rRNA reduction was performed with a RiboZero-Bacteria rRNA removal kit (Illumina, CA, USA) with the addition of custom M. barkeri MS-specific oligos designed using the sequences for M. barkeri MS’s large and small ribosomal subunits. Stranded cDNA libraries were prepared from rRNA-depleted mRNA using the TruSeq Stranded Total and mRNA kit (Illumina, CA, USA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Paired-end reads were processed using default settings in TrimGalore!, a wrapper that implements CutAdapt and FastQC to remove adapter sequences and filter reads, respectively.
Reads were aligned to the reference M. barkeri MS genome (ASM97002v1) using Bowtie2.
Reads were counted for each locus using HTSeq.
Normalization and statistical analyses were conducted in DESeq2 implemented in R v3.6.0.
Genome_build: ASM97002v1
Supplementary_files_format_and_content: All processed data files contain raw count data output from HTSeq. These data have not been normalized.
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| Submission date |
Mar 15, 2021 |
| Last update date |
Mar 13, 2022 |
| Contact name |
Rachel L. Spietz |
| E-mail(s) |
rachel.spietz@montana.edu
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| Organization name |
Montana State University
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| Street address |
PO Box 173520
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| City |
Bozeman |
| State/province |
MT |
| ZIP/Postal code |
59717 |
| Country |
USA |
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| Platform ID |
GPL29851 |
| Series (1) |
| GSE168895 |
Regulatory response of methanogen cells to different forms of iron and sulfur |
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| Relations |
| BioSample |
SAMN18310798 |
| SRA |
SRX10340484 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5172036_MbcysD.count.txt.gz |
15.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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