|
| Status |
Public on Dec 30, 2020 |
| Title |
300-LNT-1 |
| Sample type |
SRA |
| |
|
| Source name |
human infant feces
|
| Organism |
Bifidobacterium longum subsp. infantis |
| Characteristics |
strain: UMA300
treatment: lacto-N-tetraose
|
| Treatment protocol |
lactose, pooled human milk oligosaccharides (HMOs), lacto-N-tetraose (LNT), lacto-N-neotetrraose (LNnT)
|
| Growth protocol |
Bifidobacterium infantis strains were routinely propagated in De Man Rogosa Sharp medium supplemented with 0.05 % (m/v) L-cysteine hydrochloride at 37°C under anaerobic conditions. Cells then transferred into modified MRS medium (no carbohydrate source, no acetate). Carbohydrate source is replaced with either 2% w/v lactose, pooled human milk oligosaccharides, lacto-N-tetraose, lacto-N-neotetraose. The cells were grown under same conditions.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell pellets stored in RNAlater were centrifuged at 14,000 × g for 3 min and cell pellets were washed with 1 mL cold 1X PBS buffer. Total RNA was extracted using Ambion RNAqeous kit with slight modifications. Accordingly, cells were suspended in 600 μL lysis buffer in beadbeating tubes using a FastPrep 24 bead beader (MP Biomedicals, USA). Total RNA, then, were subjected to DNase treatment using the Ambion Turbo DNA-free kit. The RNA was evaluated for genomic DNA contamination using qRT-PCR. ribosomal RNA is depleted via RiboMinus kit with Magnetic Bead Clean up Module using the custom Pan-Prokaryote Probe Mix to target bacterial rRNA. Purified mRNA was used to asseses for quality and used for RNA-seq library preparation.
mRNA enriched library preparation was performed with the NEBNext Ultra II Directional kit following the manufacturer's instructions
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Sequence reads were automatically trimmed for adaptor sequence by Illumina. Adaptor trimmed reads were obtained from Illumina BaseSpace.
Reads from each lane were concatenated as left and right sequences. Left and right reads were then checked for low-quality reads, bacteria 16S, 23S and 5S rRNA contamination. B. infantis 23S rRNA region was removed using bbduk in bbmap (v38.34).
The resultant reads were concatenated to create a reference genome. The overall reads were then subject to de novo assembly using Trinity using parameters --CPU 10 --bflyCPU 6 --bflyHeapSpaceMax 2G. The statistics and the quality of the resultant transcriptome was determined by mapping the transcriptome to the raw reads using bowtie2 v2.3.4.3 using parameters -p 8 and using TrinityStats.pl function.
The estimated abundance for each sample was calculated using RSEM (v1.3.3) The quality of biological replicates and samples were determined with the PtR function in trinity and sample correlation matrix were generated using built-in pheatmap function in R (v3.6.2)
The resultant isoform count matrix was subject to differential analysis with a built-in function in Trinity using EdgeR method (25) in R (v.3.4.0) and differentially expressed genes within strains and across strains were identified paratemeter with p<0.05; log2 fold change >2, with minimum row sum of 10 raw counts)
Genome_build: Bifidobacterium longum subsp. infantis ATCC 15697
Supplementary_files_format_and_content: Matrix table with raw gene counts for every gene and every strain
Supplementary_files_format_and_content: Normalized diffferential expression measurements from edgeR
Supplementary_files_format_and_content: Transcript fasta files
|
| |
|
| Submission date |
Dec 29, 2020 |
| Last update date |
Dec 30, 2020 |
| Contact name |
Ezgi Ozcan |
| E-mail(s) |
eozcan@umass.edu
|
| Organization name |
University of Massachusetts Amherst
|
| Street address |
424 Chenoweth Lab, 102 Holdsworth Way
|
| City |
Amherst |
| State/province |
MASSACHUSETTS |
| ZIP/Postal code |
01003 |
| Country |
USA |
| |
|
| Platform ID |
GPL28926 |
| Series (1) |
| GSE164005 |
Bifidobacterium longum subsp. infantis (B. infantis) strain dependent utilization of human milk oligosaccharides |
|
| Relations |
| BioSample |
SAMN17178221 |
| SRA |
SRX9751674 |
