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| Status |
Public on Mar 24, 2023 |
| Title |
ecol_hs_r3_s_o_v17 |
| Sample type |
SRA |
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| Source name |
Liquid culture of Escherichia coli MG1655
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| Organism |
Escherichia coli |
| Characteristics |
genotype: wild type
treatment: Heat shock at 65˚C for 10 minutes
molecule subtype: total RNA, 5' monophosphorylated
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| Treatment protocol |
As for Chloramphenicol (CAM) treatment, Bacterial cells were incubated with Chloramphenicol (100µg/ml final) for 5 minutes at 37˚C with aeration and harvested at 4˚C on ice supplemented with Chloramphenicol (~100µg/ml final). Mupirocin (MUP) was added to bacterial culture at a final concentration of 65µg/ml and incubated for 10 minutes at 37˚C with aeration. Cultures for stationary growth phase samples were inoculated with single colony and harvested at indicated times post-inoculation. Salt stress samples were generated as described in PMID: 28290514 with minor modifications Salt stress was initiated by mixing equal volumes of bacterial growth culture and 2M NaCl (~5.8% NaCl). Bacterial cells were exposed to salt stress for 10 minutes at room temperature and harvested by centrifugation. Cultures for stationary growth phase samples were inoculated with single colony and harvested at indicated times post-inoculation. Heat shock experiments were setup the following. Lactobacillus plantarum (ATCC 8014) growth culture was harvested by centrifugation and heat shock was induced by resuspending cell pellet in prewarmed broth media following 15-minute incubation at 60˚C. To generate low nutrient samples, actively growing Lactobacillus plantarum (ATCC 8014) culture was harvested by centrifugation. Low nutrient stress was induced for 15 minutes beginning with excessive washing of the cell pellet with Low nutrient media (0.5x LB broth), followed by centrifugation. Washed cell pellet was resuspended in 0.5x LB broth, incubated at 37˚C for the remaining time. Polyribosome fractions were generated as described in PMID: 27146487 with minor modifications. For short, exponentially grown bacterial cells were harvested by centrifugation (5 minutes) at 4˚C on ice supplemented with 100µg/ml Chloramphenicol. Lysis was done in 1xTN buffer (50mM TRIS/HCl pH 7.4,150mM NaCl, 1mM DTT, 100μg/ml CAM and Complete EDTA Free Protease inhibitor tablet) using glass beads and of vortexing (2minutes) followed by resting of sample on ice (5 minutes). Vortexing/resting cycle was repeated two more times. Lysate was cleared by centrifugation at 1500g for 5 minutes at 4˚C. Resulting supernatant was loaded on to a 15-50% sucrose gradient with a 80% cushion and sedimentation was accomplished by ultracentrifugation for 90 minutes at 36,000rpm. Fractions were collected and A254 value was monitored. Subsequently, RNA was extracted from sucrose fractions by adding equal volumes of phenol/chloroform isoamyl alcohol pH 4.5 (25:24:1) and nuclease free water followed by 2-minute vortexing and centrifugation. Aqueous phase was further cleaned up by the addition of Chloroform, vortexing and centrifugation. Resulting aqueous phase was sodium acetate-ethanol precipitated.
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| Growth protocol |
Bacteria were grown according to their requirements. Overnight Cultures were diluted to OD600 0.01-0.07, growth was continued and cells were harvested in log-phase by centrifugation. Resulting pellet was shock-frozen in Dry ice/Ethanol bath and stored for RNA analysis
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| Extracted molecule |
total RNA |
| Extraction protocol |
Microbial cell pellets were resuspended in equal volume of LET (25 mM Tris pH 8.0, 100 mM LiCl, 20 mM EDTA) and water saturated Phenol pH 6.6 (Thermo Fisher), lysed with acid washed glass beads (Sigma-Aldrich) and vortexing for three minutes in MultiMixer. Lysates were supplemented with equal volumes of phenol/chloroform isoamyl alcohol pH 4.5 (25:24:1) and nuclease free water, l two-minute vortexing followed by centrifugation. Resulting aqueous phase was purified in two steps using phenol-chloroform isoamyl alcohol (25:24:1) followed by chloroform. After centrifugation, the clean aqueous phase was precipitated with sodium acetate-ethanol.
Standard and random fragmented 5PSeq libraries were generated as described in PMID: 26046441 with minor modifications using 150-9000ng total RNA as an input. To prepare random fragmented samples (negative controls), ribosomal RNA was depleted from DNA-free RNA and subsequent fragmented by incubating five minutes at 80 ̊C in fragmentation buffer (40mM Tris Acetate pH 8.1, 100mM KOAc and 30mM MgOAc). Reaction was purified using 2 volumes of RNACleanXP beads (Beckman Coulter) as recommended by the manufacturer. Free 5’OH sites were re-phosphorylated using 5 Units of T4 Polynucleotide kinase (PNK, NEB) and incubated at 37 ̊C for 60 minutes as recommended by the manufacturer. Re-phosphorylated fragmented RNA was purified using Phenol:Chloroform: Isoamyl Alcohol (24:25:1), followed by sodium acetate-ethanol precipitation. From this step forward, procedures for random fragmented and standard 5PSeq library preparation merge. RNA was Ligated to either rP5_RND or rP5_RNA oligo (specified in characteristics protocol). containing unique molecular identifiers). Ribosomal RNA was depleted using Ribozero rRNA removal kit (Illumina) suitable for Bacteria and Yeast. rRNA depleted sample was purified using 1.8V of Ampure beads (Abcam), fragmented using heat (80˚C) for 5 min in 5x Fragmentation Buffer (200mM Tris Acetate pH 8.1,500mM KOAc,150mM MgOAc) and reverse transcribed using random hexamers to prime. Resulting cDNA was bound to streptavidin beads (M-280), subjected to enzymatic reactions of DNA end repair, fill-in of adenine to 5’ protruding ends of DNA fragments using Klenow Fragment (NEB). Common adaptor (P7-MPX) was ligated and 5PSeq Libraries were amplified by PCR (15- 17 cycles). Purified samples were quantified using Qubit (Thermo Fisher), library size was estimated from bioanalyzer traces. 5PSeq Libraries were pooled by mixing equal amounts of each sample, following enrichment of fragments of the length between 300-500nt. HT-5PSeq Libraries were generated as recently described (bioRxiv 2020.06.22.165134; doi: https://doi.org/10.1101/2020.06.22.165134). In brief, DNA-free RNA was ligated with RNA oligos containing unique molecular identifiers. Ligated RNA was reverse transcribed priming with oligos containing a random hexamer and an Illumina compatible region. RNA was eliminated by addition of NaOH. Ribosomal RNA was depleted by adding in-house rRNA DNA oligo depletion mixes (table 2) to the cDNA and performing a duplex-specific nuclease (DSN, Evrogen) treatment. rRNA depleted cDNA was PCR amplified (15-17 cycles). Depletion of ribosomal RNA with Ribozero Illumina (for bacteria and yeast) was done after the single-stranded RNA ligation step. Ribosomal depleted RNA was purified and reverse transcribed using the same oligos as stated above, and then amplified by PCR. Libraries were quantified by fluorescence (Qubit, Thermo Fisher), size estimated using an Agilent Bioanalyzer and sequence using a NextSeq500 Illumina sequencer.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Description |
Culture: ecol_hs_r3_s_o_v17 was cultured in LB Broth at 37˚C to log growth phase, 2 ml of culture was centrifuged and pellet was resuspended in 65˚C pr-warmed LB, incubated for 10 minutes at 65˚C on Heatblock and harvested by centrifugation; ; Library: HT-5PSeq using indexed rP5_RNA (rP5_MPX_RNA) Adapters (CrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrU rXrXrXrXrXrX rNrNrNrNrNrNrNrN) carrying a sample barcode (rX) and unique molecular identifiers (rN) which were removed from the reads into the read headers. rRNA removal: in-house Bacterial rRNA Depletion MIX for Cultures , oVP670
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| Data processing |
library strategy: 5PSeq that captures transcripts with free 5' phospahte (without cap)
Adapter and quality trimming with the bbduk program of the BBTools suite, with options {qtrim=r, ktrim=r, hdist=3, hdist2=2, K=20, mink=14, trimq=16, minlen=30, maq=16}, using BBTools default adapter set and polyG or polyA sequences for short reads.
Deduplication of reads with both identical unique molecular identifier (UMI) and insert sequence using the dedupe program of the BBTools suite with its default parameters.
UMI sequences found in the first eight bases of each read were extracted using UMI-tools (version 1) with default options (using --bc-pattern NNNNNNNN).
Dual index fastq samples were processed as described in supplementary file dual_index_demultiplexing.txt
Alignment was performed with the bbmap program of the BBTools suite, with the {32bit=t -da -eoom k=11 strictmaxindel=10 intronlen=0 t=16 trd=t minid=0.94 nzo=t} parameters. Alignment files were sorted and indexed with SAMtools.
Deduplication based on UMIs was then performed with UMI-tools (version 1) with options { --soft-clip-threshold 1 --edit-distance 2 --method unique}.
In selected samples, a dual undex strategy was used (sequencing runs v17). In brief, in addition of the Illumina indexes with dedicated reads used in HT-5Pseq, an additional index sequence is added before the UMI sequence. This is introduced during the ssRNA ligation space using anoligo with the following structure Illumina compatible-CCCCCC-NNNNNNNN. Please see dual_index_demultiplexing.txt for details on demultiplexing strategy for those samples.
Bedgraphs for read 5' positions for forward (fwd) and reverse (rev) strands were generated with BEDTools genomecov program with options {-bg -strand [+/-] -5}.
Assembly: The genome used for alignment is indicated in the processed file names with the G-xxxx patterns. Those correspond to the NCBI accessions and builds indicated in the Supplementary file.
Supplementary files format and content: Reference indiced for individual bacterial species and custom mixtures were built with the bbmap program of the BBTools suite, with default options (and k=10).
Supplementary files format and content: Processed files are bedgraphs containing read 5' counts on reverse (_rev) and forward (_fwd) strands of genomes indicated with _G-xxxx pattern. The genomes corresponding to each pattern are described in the genome build entry above.
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| Submission date |
Feb 07, 2023 |
| Last update date |
Mar 25, 2023 |
| Contact name |
Vicent Pelechano |
| E-mail(s) |
vicente.pelechano.garcia@ki.se
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| Organization name |
ScilifeLab - Karolinska Institutet
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| Department |
MTC
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| Street address |
Nobels väg 16
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| City |
Solna |
| ZIP/Postal code |
SE-17177 |
| Country |
Sweden |
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| Platform ID |
GPL32081 |
| Series (1) |
| GSE153497 |
RNA degradation analysis reveals ribosome dynamics in complex microbiome samples |
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| Relations |
| SRA |
SRX19302927 |
| BioSample |
SAMN33246954 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7030350_ecol_hs_r3_s_o_v17_G-ecol_fwd.bedgraph.gz |
35.8 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM7030350_ecol_hs_r3_s_o_v17_G-ecol_rev.bedgraph.gz |
32.9 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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