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| Status |
Public on Aug 20, 2013 |
| Title |
Pul 1 |
| Sample type |
SRA |
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| Source name |
In vitro bacterial culture, Pullulan from Aureobasidium pullulans
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| Organism |
Bacteroides cellulosilyticus |
| Characteristics |
strain: WH2
experiment: NM602
carbohydrate: Pullulan from Aureobasidium pullulans
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| Treatment protocol |
Sample was collected from an in vitro culture in which Bacteroides cellulosilyticus WH2 was grown to early log stage in Bacteroidetes minimal media containing a single carbohydrate as a carbon source.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNAprotect Bacteria Reagent (Qiagen) was used to stabilize both frozen fecal pellets (experiment NM601) and in vitro cultures (experiment NM602; added prior to pelleting cells) per the manufacturer's instructions. Samples were then mechanically disrupted using acid-washed glass beads in a Mini-BeadBeater-8 in the presence of acid phenol:chloroform:IAA (125:24:1), after which ethanol precipitation was performed. RNA was then further purified using a MEGAclear kit, tested for integrity by gel electrophoresis, and shown to be lacking any contaminating genomic DNA by PCR. rRNA depletion was subsequently performed via two rounds of processing with the MICROBExpress kit and two rounds of rRNA capture using custom oligos attached to magnetic beads (Dynabeads). First-strand cDNA synthesis was then performed using random hexamer priming with SuperScript II, after which second-strand synthesis was achieved using E. coli DNA polymerase in the presence of RNase H.
Libraries were prepared according to 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. Non-barcoded adapters were used to construct libraries from experiment NM601 samples. Adapters incorporating an 8 bp barcode were used to construct libraries from experiment NM602 samples. 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 either the Illumina Genome Analyzer IIx (experiment NM601) or the Illumina HiSeq 2000 (experiment NM602) using the manufacturer's protocols.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
NM602-F_82_Pul_H-4
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| Data processing |
Basecalls performed using RTA (experiment NM601, v1.5.35-1.6.47; experiment NM602 v1.13.48).
PhiX reads from spike-in controls were filtered out by aligning all reads to a PhiX reference using Bowtie with default settings.
For experiment NM602, multiplexed sequences were demultiplexed based on a perfect match to each 8 bp barcode used, and barcodes were trimmed.
Reads were aligned to one (experiment NM602) or multiple (experiment NM601) reference genomes using the SSAHA2 aligner.
In some cases, reads mapped non-uniquely (i.e., to multiple locations within or across reference genomes). To account for this, we first tallied read counts by gene for all uniquely mapping sequences. Subsequently, reads that mapped non-uniquely were added to each gene's read count in proportion to each gene's fracton of unique match counts. We also added a pseudocount (i.e., a value of 1) to each gene count prior to normalization to account for differences in sampling depth. Read counts were then subsequently normalized at either a species level (i.e., taking into account all genes for a single species) or at a community level (i.e., taking into account all genes for all species) using DESeq.
Genome_build: AAVM00000000.2, AAXF00000000.2, AE015928.1, AAYH00000000.2, CP000139.1, ABFY00000000.2, ABIK00000000.2, AAVN00000000.2, AAXB00000000.2, CP000140.1, AAVO00000000.2; note that one additional genome included in the analysis (that of B. cellulosilyticus WH2) was sequenced as part of this study and has not yet been assigned an NCBI accession number.
Supplementary_files_format_and_content: Tab-delimited text file. Each file contains four columns of information. Column 1 (feature_name): gene. Column 2 (hits): raw counts attributed to gene. Column 3 (fraction_of_reads): proportion of all reads mapped assigned to gene. Column 4 (average_match_score): average match score reported by the SSAHA2 aligner for all reads assigned to gene.
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| Submission date |
Jul 17, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Nathan P McNulty |
| E-mail(s) |
[email protected]
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| Phone |
314-362-3963
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| Organization name |
Washington University School of Medicine
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| Department |
Center for Genome Sciences and Systems Biology
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| Lab |
Gordon
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| Street address |
4444 Forest Park Ave. (5th Floor)
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| City |
Saint Louis |
| State/province |
MO |
| ZIP/Postal code |
63108 |
| Country |
USA |
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| Platform ID |
GPL17468 |
| Series (2) |
| GSE48537 |
Effects of Diet on Resource Utilization by a Model Human Gut Microbiota Containing Bacteroides cellulosilyticus WH2, a Symbiont with an Extensive Glycobiome |
| GSE48993 |
Effects of Diet on Resource Utilization by a Model Human Gut Microbiota Containing Bacteroides cellulosilyticus WH2, a Symbiont with an Extensive Glycobiome (RNA-Seq) |
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| Relations |
| BioSample |
SAMN02259033 |
| SRA |
SRX326359 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1191877_NM602-F_82_Pul_H-4.counts.txt.gz |
42.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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