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| Status |
Public on Nov 09, 2018 |
| Title |
C57BL/6 Gut Microbiome with Low Fat Diet, DM, Mouse3 |
| Sample type |
SRA |
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| Source name |
C57BL/6 (Gut Microbiome)
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| Organism |
mouse gut metagenome |
| Characteristics |
diet: low fat
tissue: mouse cecum
treatment: demethylase
host: Mus musculus (mouse)
host strain: C57BL/6
molecule subtype: tRNA
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| Treatment protocol |
Sequenced tRNA was derived from gut micrbiome from age and litter-matched specific pathogen free (SPF) or germ free (GF) male C57Bl/6 mice between 8-12 weeks old fed either a purified low fat (LF; Harlan Teklad TD.00102) diet, high saturated milk fat (MF; Harlan Teklad TD.97222) diet, or high polyunsaturated fat (PUFA; Harlan Teklad TD.97223) diet for four weeks ad libitum.
DM refers to demethylase treatment. This is a cocktail of AlkB and AlkB engineered mutants that remove modifications from the tRNAs. This treatment was performed after isolation of tRNA from mouse cecum.
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| Growth protocol |
We bred C57BL/6 mice and maintained them under standard 12:12 h light/dark conditions at the University of Chicago. We fed age and litter-matched specific pathogen free (SPF) or germ free (GF) male C57Bl/6 mice between 8-12 weeks old either a purified low fat (LF; Harlan Teklad TD.00102) diet, high saturated milk fat (MF; Harlan Teklad TD.97222) diet, or high polyunsaturated fat (PUFA; Harlan Teklad TD.97223) diet for four weeks ad libitum. Diets were formulated by Harlan Teklad and compositions were published previously15. We γ-irradiated gnotobiotic diets and tested them for sterility prior to use. We anesthetized mice using 10 mg/ml ketamine/xylazine followed by exsanguination and cervical dislocation. We snap-froze cecal contents in liquid nitrogen and stored at -80°C.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cecal contents were snap-frozen in liquid nitrogen and stored at -80°C. Cecal contents were lysed in 400μL of 0.3M NaOAc/HOAc, 10mM EDTA pH 4.8 with an equal volume acetate saturated phenol chloroform pH 4.8 and were placed in a reciprocating bead beater on max using for 45 seconds using FastPrep lysing matrix B. Purified total RNA was deacylated by incubating in 0.1 M tris-HCl, pH 9 at 37°C for 45 min. Total tRNA was subsequently isolated using a denaturing 10% polyacrylamide gel followed by passive gel elution and ethanol precipitation.
Homemade protocol based on Illumina protocols. Deacylated RNAs with or without the demethylation were first treated with T4 Polynucleotide Kinase (Epicentre) at 37C° for 30 min to further warrant a free 3' hydroxyl group for template switching. Template-switching reactions were performed as described7. Briefly, we used an initial template-primer substrate consisting of a 41-nt RNA oligonucleotide (5’-AGA UCG GAA GAG CAC ACG UCU AGU UCU ACA GUC CGA CGA UC/3SpC3/-3’) that contains Illumina Read1 and Read2 primer-binding sites and a 3’ blocking group (three carbon spacer; IDT) annealed to a complementary 32P-labeled DNA primer with a single-nucleotide 3’ overhang, T, which facilitates the template switch to full-length tRNA that mostly contain a 3’ CCA end. For g2RT template-switching reactions, average 100 ng of demethylated tRNAs or 1 μg of demethylated total RNA were mixed with the initial template-primer substrate (100 nM) and g2RT (1 μM of TeI4cΔEn or 500 nM of GsI-IIC RT) in reaction medium containing 450 mM NaCl, 5 mM MgCl2, 20 mM Tris-HCl, pH 7.5, and DTT (1 mM for TeI4cΔEn or 5 mM for GsI-IIC RT). For template-switching with TeI4cΔEn RT, the reactions were pre-incubated at room temperature for 30 min, initiated by adding 25 mM dNTPs to a final concentration of 1 mM and incubating at 60°C for 30 min. For template-switching with GsI-IIC RT, the pre-incubation step is not required. The reactions were terminated by adding 5 M NaOH to a final concentration of 0.25 M, incubating at 95°C for 3 min, and neutralizing with 5M HCl. The cDNAs resulting from template switching were analyzed in a denaturing 6% polyacrylamide gel, electroeluted using a D-tube Dialyzer Maxi with MWCO of 6-8 kDa (EMD Millipore), and ethanol precipitated with 0.3 M sodium acetate in the presence of 25 µg of linear acrylamide (Life Technologies). The purified cDNAs were then circularized with CircLigase II (Epicentre) using manufacture’s protocol with extended incubation time of 5 hours at 60°C, phenol-CIA extracted, ethanol precipitated and amplified with Phusion-HF (Thermo Scientific) using Illumina multiplex (5’- AAT GAT ACG GCG ACC ACC GAG ATC TAC ACG TTC AGA GTT CTA CAG TCC GAC GAT C -3’) and barcode (5’- CAA GCA GAA GAC GGC ATA CGA GAT BARCODE GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T -3’) primers for 12 cycles of 98°C for 5 sec, 60°C for 10 sec and 72°C for 10 sec. The PCR products were sequenced on Illumina HiSeq 2000 or Illumina MiSeq system.
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| Library strategy |
ncRNA-Seq |
| Library source |
transcriptomic |
| Library selection |
size fractionation |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Low fat mouse, biological replicate 3, which had tRNA subsequently isolated
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| Data processing |
We removed low-quality reads from the raw sequencing results using illumina-utils 17 (available from https://github.com/merenlab/illumina-utils). For the analysis of 16S rRNA gene amplicons data we used the program ‘iu-merge-pairs’ with default parameters, which merged partially overlapping paired-end Illumina reads while simultaneously removing any pair with more than 3 mismatches at the overlapped region. For pairs with three or less mismatches, we picked the base to be used in the final merged sequence from the read with the higher Q-score
We used Minimum Entropy Decomposition 11 with default parameters to cluster high-quality 16S rRNA gene amplicons at 1-nt resolution, and GAST 18 to assign taxonomy to each read individually.
To recover tRNA sequences in this dataset we also used the program ‘iu-merge-pairs’, but with two additional flags and an additional parameter: --retain-only-overlap (to keep only the overlapping region while trimming tailing ends from both reads in a pair), --marker-gene-stringent (to align the reverse-complement of the second read even if the alignment occurs between the end of the second read and the beginning of the first read --which is a unique case for short inserts), and --max-num-mismatches 0 (to remove any pair if there was a disagreement between the aligned portions to increase the quality dramatically as described in 17)
We developed a new software tool to identify tRNA sequences in the raw sequencing results: tRNA-seq-tools (available from https://github.com/merenlab/tRNA-seq-tools). We used the tRNA-seq-tools program ‘trna-profile’ with default parameters to identify all sequences that matched our criteria for standard tRNA
Supplementary_files_format_and_content: Mapped tRNA sequences in text files as output from tRNA seq tools.
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| Submission date |
Jun 20, 2017 |
| Last update date |
Nov 10, 2018 |
| Contact name |
Tao Pan |
| E-mail(s) |
taopan@uchicago.edu
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| Phone |
(773) 702-4179
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| Organization name |
University of Chicago
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| Department |
Biochemistry and Molecular Biology
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| Street address |
929 E. 57th Street
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| City |
Chicago |
| State/province |
Illinois |
| ZIP/Postal code |
60637 |
| Country |
USA |
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| Platform ID |
GPL17443 |
| Series (1) |
| GSE100263 |
Microbiome characterization using transfer RNA sequencing |
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| Relations |
| BioSample |
SAMN07258600 |
| SRA |
SRX2937115 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2676381_tRNA-DM-LF-M03-tRNAs.map.txt.gz |
8.9 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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