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| Status |
Public on Jun 07, 2020 |
| Title |
ATCC2FLB |
| Sample type |
SRA |
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| Source name |
Bacterial culture
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| Organism |
Bifidobacterium longum subsp. infantis |
| Characteristics |
strain: ATCC 15697
carbon source: 2’-fucosyllactose
growth phase: Mid-log
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| Treatment protocol |
Carbon source
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| Growth protocol |
For all experiments, we modified the basal media from Asakuma et al. 8 to create modified basal media (mBasal) containing: 10 g/L BD peptone, 2 g/L yeast extract, 5 g/L NaCl, 2 g/L diammonium citrate, 0.2 g/L magnesium sulfate, 2 g/L dipotassium hydrogen phosphate. The pH was adjusted to 6.4. After autoclaving, we added filter-sterilized (0.2uM, PES membrane) 0.05% (w/v) L-cysteine HCl and 1% (w/v) of the indicated carbon source.
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
After each sample reached its desired A600 range, cells were centrifuged for 10 min. at 4,063 x g. The supernatant was removed and 1 mL of TRIzol (p/n: 15596026, Thermo) was added. The pellet was re-suspended by vortexing and the tubes were immediately frozen at -80°C. The cell pellets were later thawed, transferred to a Lysing Matrix B 2mL tube (p/n: 116911050, MPBio, Santa Ana, CA), and disrupted using a Mini-Beadbeater. The lysate was subjected to a chloroform organic extraction and followed by purification using RNeasy Mini Kit (p/n: 74104, Qiagen, Hilden, Germany).
Quality checks and quantifications of the isolated RNA were carried out on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). Ribosomal RNA was removed prior to library construction using Ribo-Zero rRNA Removal Kit Gram-Positive Bacteria (p/n: MRZGP126, Illumina). Stranded cDNA libraries were prepared using TruSeq Stranded mRNA Kit (p/n: 20020594, Illumina), quantitated by Agilent TapeStation, pooled quimolarly, and sequenced on one flowcell lane for 75 cycles using paired-end 75 basepair sequencing on Illumina 2500 HiSeq Rapid Cluster Kit v. 2 (p/n: PE-402-4002, Illumina) and HiSeq Rapid SBS Kit v. 2 (p/n: FC-402-4021, Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
A600= 0.5-0.9
ATCC2FLB
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| Data processing |
Paired-end reads were imported and mapped to the B. longum subsp. infantis Bi-26 genome (SAMN10380491) using the “Geneious for RNA” Mapper with default settings in Geneious v. 11.0.4
Transcript levels in the resulting assemblies were calculated using the “Calculate Expression Levels” function, which enabled the comparison of replicates and experiments using the “Compare Expression Levels” with the DESeq2 method and parametric fit type.
The assemblies were exported as BAM files and imported into ArrayStar v. 1.2 (DNAStar, Madison, WI), processed using QSeq, and normalized by reads per kilobase of transcript per million mapped reads (RPKM).
Regression analyses of the RNA data were made in ArrayStar software using the Student’s t-test with FDR correction. Statistical analyses between sets of samples were analyzed using DESeq2 method as above. Differences in expression were considered significant if the Absolute Confidence (-Log10 adjusted p-value) was +1.00, and the Log2 ratio was at + 1.00, representing p < 0.05 and a > 2x fold change, respectively, after normalization.
Genome_build: RJJM00000000
Genome_build: NC_011593
Supplementary_files_format_and_content: gene count tables and alignment using Geneious Prime v. 2020.0.5
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| Submission date |
Jun 06, 2020 |
| Last update date |
Jun 07, 2020 |
| Contact name |
Bryan Zabel |
| E-mail(s) |
bryan.zabel@iff.com
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| Organization name |
International Flavors & Fragrances Inc
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| Department |
GEM
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| Street address |
3329 Agriculture Dr
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| City |
Madison |
| State/province |
wi |
| ZIP/Postal code |
53716 |
| Country |
USA |
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| Platform ID |
GPL25787 |
| Series (1) |
| GSE151933 |
Strain-specific strategies of 2′-fucosyllactose, 3-fucosyllactose, and difucosyllactose assimilation by Bifidobacterium longum subsp. infantis Bi-26 and ATCC 15697 |
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| Relations |
| BioSample |
SAMN15154027 |
| SRA |
SRX8485034 |
| 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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