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Sample GSM3258510

Query DataSets for GSM3258510

Status

Public on Nov 09, 2018

Title

Staphylococcus aureus, DM, replicate 3

Sample type

SRA

Source name

bacterial culture

Organism

Staphylococcus aureus

Characteristics

treatment: demethylase
tissue: bacterial culture
molecule subtype: tRNA

Treatment protocol

Sequenced tRNA was derived from bacterial pellets. 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.

Growth protocol

We grew E. coli, B. subtilis, and S. aureus cultures at 37°C in Erlenmeyer flasks with a 10:1 flask medium ratio at 225rpm. We grew E. coli and B. subtilis in LB Lennox and S. aureus in tryptic soy broth. We grew B. viscericola in serum vials under strict anaerobic conditions at 37°C in chopped meat medium as previously described1. We harvested all cultures at midlog by centrifugation.

Extracted molecule

total RNA

Extraction protocol

We lysed pelleted bacteria in 400μL of 0.3M NaOAc/HOAc, 10mM EDTA pH 4.8 with an equal volume acetate saturated phenol chloroform pH 4.8. We placed samples in a reciprocating bead beater for 45 seconds on maximum intensity using FastPrep lysing matrix B. We centrifuged samples at 21,000rcf for 15 minutes at 4°C before re-extraction and ethanol precipitation of total RNA.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 system.

Library strategy

ncRNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2000

Description

processed data file : StaphAureusCount.xlsx
Staphylococcus aureus, biological replicate 3, demethylase treated, which had tRNA subsequently isolated

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: Bacterial tRNA counts in Excel file format

Submission date

Jul 05, 2018

Last update date

Nov 09, 2018

Contact name

Tao Pan

E-mail(s)

taopan@uchicago.edu

Phone

(773) 702-4179

Organization name

University of Chicago