|
| Status |
Public on Nov 01, 2011 |
| Title |
in vitro culture NM1200_InVitro_5_E3t2 |
| Sample type |
SRA |
| |
|
| Source name |
in vitro culture
|
| Organisms |
Lactobacillus delbrueckii subsp. bulgaricus; Lactococcus lactis subsp. lactis CNCM I-1631; Bifidobacterium animalis subsp. lactis CNCM I-2494; Streptococcus thermophilus CNCM I-1630 |
| Characteristics |
sample type: milk fermentation reaction
source: a milk fermentation reaction 3 hours after inoculation of the milk. The milk in these reactions included a B. animalis subsp. lactis growth-enhancing ingredient. B. animalis subsp. lactis represents approximately 44% of the total CFUs in these samples. Biological replicate #2 of 3.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Libraries were prepared according a slightly modified version of the protocol accompanying the Illumina Genomic DNA Sample Prep Kit. Briefly, cDNA was sonicated in a BioRuptor XL water bath sonicator, cleaned up and concentrated using a Qiagen PCR Purification column, and end-repaired using Klenow DNA polymerase. The blunt DNA was treated with Klenow fragment (exo minus) to add an adenine overhang, and the A-tailed molecules were ligated to the relevant Illumina adapter sequence (with or without a 4-nt barcode). Adaptered-DNA was then size-selected by agarose gel electrophoresis. Fragments of the appropriate size were PCR amplified and purified, after which the purified PCR products were loaded on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer (IIx) following the manufacturer's protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
Bifidobacterium animalis subsp. lactis CNCM I-2494,Lactobacillus delbrueckii subsp. bulgaricus CNCM I-1632,Lactobacillus delbrueckii subsp. bulgaricus CNCM I-1519,Lactococcus lactis subsp. cremoris CNCM I-1631,Streptococcus thermophilus CNCM I-1634
rRNA-depleted total RNA
RNA isolated from a milk fermentation reaction 3 hours after inoculation of the milk. The milk in these reactions included a B. animalis subsp. lactis growth-enhancing ingredient. B. animalis subsp. lactis represents approximately 44% of the total CFUs in these samples. Biological replicate #2 of 3.
|
| Data processing |
After parsing each sequencing lane's data by barcode (where appropriate), we excised the barcodes and mapped the reads to an appropriate set of reference genomes using the ssaha2 algorithm. Minimum score thresholds for ssaha were selected based on the distribution of scores for all mapped reads of a 32-nt barcoded sample and a 36-nt non-barcoded sample (29 was selected as the minimum score for 32-nt barcoded samples; 33 was the minimum score used for 36-nt and 76-nt non-barcoded samples). Although an 18-nt read is sufficient to map more than 90% of the sequencing reads, even at 32-36 nucleotides a large fraction of reads map to multiple locations (within a genome and/or across genomes). Reads that map non-uniquely were added to each gene in proportion to each gene's fraction of unique-match counts (e.g., a non-unique read that maps equally well to gene A with 18 unique reads and gene B with 2 unique reads will result in 0.9 counts being tallied to gene A and 0.1 counts being tallied to gene B). We added a pseudocount (i.e. added a value of 1) to each gene count prior to normalization to account for differences in sampling depth, although clearly a more appropriate model-based approach to smooth lower expression values is needed in the future, particularly with mixed species samples where one or more species is clearly undersampled. Raw counts were ultimately normalized to reads/kb gene length/million mapped reads (RPKM) following gene count assignments.
|
| |
|
| Submission date |
Aug 25, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Nathan P McNulty |
| E-mail(s) |
nathan.p.mcnulty@gmail.com
|
| Phone |
314-362-3963
|
| Organization name |
Washington University School of Medicine
|
| Department |
Center for Genome Sciences and Systems Biology
|
| Lab |
Gordon
|
| Street address |
4444 Forest Park Ave. (5th Floor)
|
| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63108 |
| Country |
USA |
| |
|
| Platform ID |
GPL14350 |
| Series (1) |
| GSE31670 |
The impact of a consortium of fermented milk strains on the gut microbiome of gnotobiotic mice and monozygotic twins. (RNA-Seq) |
|
| Relations |
| SRA |
SRX093154 |
| BioSample |
SAMN00714051 |
