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| Status |
Public on Apr 23, 2019 |
| Title |
bi26lacmid_01 |
| Sample type |
SRA |
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| Source name |
Bacterial culture
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| Organism |
Bifidobacterium longum subsp. infantis |
| Characteristics |
strain: Bi-26
carbon source: Lactose
growth phase: Mid-log
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| Treatment protocol |
Carbon source
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| Growth protocol |
Samples grown anerobicall in modified Bifidobacterium media 58 (mBM58) at 38°C. mBM58 from Deutsche Sammlung von Microorganismen und Zellculturen; [casein peptone, tryptic digest 10 g/L, yeast extract 5 g/L, meat extract 5 g/L, K2HPO4 3 g/L, ascorbic acid 10 g/L, and cysteine-HCl 0.5 g/L; pH 6.8].
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| Extracted molecule |
total 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
raw_rnaseq_counts.tsv
Bi26_2'FL_fullcomps.gff
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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
Supplementary_files_format_and_content: .bam sequence alignment using Geneious v. 11.0.4
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| Submission date |
Nov 08, 2018 |
| Last update date |
Apr 23, 2019 |
| Contact name |
Wesley w. Morovic |
| E-mail(s) |
wesley.morovic@dupont.com
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| Phone |
608-395-2819
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| Organization name |
DuPont Nutrition & Health
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| Department |
Genomics & Microbiome Science
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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) |
| GSE122350 |
Novel Genes and Metabolite Trends in Bifidobacterium longum ssp. infantis Bi-26 Involved in Metabolism of Human Milk Oligosaccharide 2’-fucosyllactose |
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| Relations |
| BioSample |
SAMN10406654 |
| SRA |
SRX4999988 |
| 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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