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| Status |
Public on Jun 11, 2020 |
| Title |
WT TEX rep1 MLP |
| Sample type |
SRA |
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| Source name |
cultures
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| Organism |
Bacteroides thetaiotaomicron |
| Characteristics |
strain: VPI-5482
genotype: wild-type
phase: mid-logarithmic
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| Growth protocol |
Samples 1-18: Wild-type B. thetaiotaomicron VPI-5482 was grown overnight in 5 mL pre-reduced TYG medium followed by sub-culturing (1:100) into 100 mL fresh pre-reduced TYG. Total RNA was extracted from culture aliquots of three biological replicates at early-exponential (4 h), mid-exponential (7 h), and stationary growth phases (10 h). Samples 19-24: B. thetaiotaomicron wild-type (tdk mutant background), ∆BTnc035, (BTnc035 deletion mutant), and BTnc035+ (BTnc035 complementation strain) strains were grown in TYG to stationary phase, followed by the addition of anhydrotetracycline at a final concentration of 200 ng/mL, and resumed growth therein for 2 h.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by hot phenol extraction from 4 OD equivalents of culture to which 1.6 mL stop mix (Eriksson et al., 2003) was added (95% v/v ethanol, 5% v/v water saturated phenol, pH >7.0). The cells were lysed by incubation with 600 μL lysozyme (0.5 mg/mL) and 60 μL 10% SDS for 2 min at 64°C, followed by the addition of 66 μL 3 M NaOAc. Phenol extraction (750 μL; Roti-Aqua phenol) was performed at 64°C for 6 min with the subsequent addition of 750 µL of chloroform. RNA was precipitated from the aqueous phase with twice the volume of 30:1 (ethanol : 3 M NaOAc, pH 6.5) mix and incubated at –80°C overnight. After centrifugation, pellets were washed with 75% (vol/vol) ethanol and re-suspended in 50 μL H2O. Contaminating genomic DNA was removed by treating 40 μg total RNA with 5 U of DNase I (Fermentas) and 0.5 μL Superase-In RNase Inhibitor (Ambion) in a 50 μL reaction (1 U/µL, Fermentas). RNA quality was checked using a 2100 Bioanalyzer and the RNA 6000 Nano kit (Agilent Technologies). RNA integrity (RIN) values for all samples were >7.
Samples 1-18: Prior to cDNA synthesis, total RNA was fragmented using ultrasound (4 pulses of 30 sec at 4°C) and treated with T4 Polynucleotide Kinase. Subsequently, half the total RNA of each sample was treated with Terminator exonuclease (TEX) to enrich for primary transcripts. RNA samples were then poly (A)-tailed using poly (A) polymerase and 5’-PPP was removed with 5’ Polyphosphatase. RNA adaptors were ligated and first-strand cDNA synthesis was carried out using oligo (dT) primers and M-MLV reverse transcriptase. The cDNA was PCR amplified to about 10-20 ng/μL, purified using Agencourt AMPure XP kit and fractionated in a size range of 200-500 bp. Samples 19-24: cDNA libraries were prepared using the NEBNext Multiplex Small RNA Library Prep kit for Illumina in accordance with the manufacturers’ instructions with modifications: RNA samples were fragmented with Mg2+ at 94˚C for 2.75 min using the NEBNext Magnesium RNA Fragmentation Module followed by RNA purification with the Zymo RNA Clean & Concentrator kit. Fragmented RNA was dephosphorylated at the 3’ end, phosphorylated at the 5’ end and decapped using 10 U T4-PNK +/- 40 nmol ATP and 5 U RppH, respectively. After each enzymatic treatment, RNA was purified with the Zymo RNA Clean & Concentrator kit. The small RNA fragments were ligated for cDNA synthesis to 3’ SR adapter and 5’ SR adapter diluted 1:3 with nuclease-free water before use. PCR amplification to add Illumina adaptors and indices to the cDNA was performed for 14 cycles with 1:3 diluted primer. Barcoded DNA libraries were purified using magnetic MagSi-NGSPREP Plus beads at a 1.8 ratio of beads to sample volume.
Samples 1-18: dRNA-seq; samples 19-24: RNA-seq
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Terminator exonuclease (TEX) treated
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| Data processing |
Basecalling: Illumina version 2.2.0
Read trimming: Cutadapt version 2.5 including –nextseq-trim=20 switch to handle two-color sequencing.
Reads were mapped to the B. thetaiotaomicron VPI-5482 genome and plasmid using the READemption pipeline that employs the Segemehl algorithm for split reads.
Genome_build: NC_004663.1 (chromosome), NC_004703.1 (plasmid)
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| Submission date |
Jan 29, 2020 |
| Last update date |
Jun 11, 2020 |
| Contact name |
Lars Barquist |
| E-mail(s) |
lars.barquist@helmholtz-hiri.de
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| Organization name |
Helmholtz Institute for RNA-based Infection Research
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| Street address |
Josef-Schneider-Straße 2 / Bau D15
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| City |
Würzburg |
| State/province |
Bayern |
| ZIP/Postal code |
97080 |
| Country |
Germany |
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| Platform ID |
GPL28094 |
| Series (1) |
| GSE144492 |
High-resolution view at the coding transcriptome and noncoding RNAs of major gut microbe Bacteroides thetaiotaomicron |
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| Relations |
| BioSample |
SAMN13941626 |
| SRA |
SRX7648636 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4289294_L1801823_S4_Tex_p1_forward.wig.gz |
4.1 Mb |
(ftp)(http) |
WIG |
| GSM4289294_L1801823_S4_Tex_p1_reverse.wig.gz |
4.4 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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