5'-specific RNA-Seq of Corynebacterium glutamicum wildtype, deletion mutant sigE and deletion mutant cseE reveals TSS and promoters depending on SigE and thereby overlapping SigH and SigE sigma factor regulons.
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ABSTRACT:
INSTRUMENT(S): Illumina MiSeq
ORGANISM(S): Corynebacterium glutamicum
SUBMITTER:
PROVIDER: E-MTAB-12458 | biostudies-arrayexpress |
SECONDARY ACCESSION(S): E-MTAB-12457
REPOSITORIES: biostudies-arrayexpress
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Project description:Total RNA was isolated from 3 biological replicates of C. glutamicum wildtype, deletion mutant sigE and deletion mutant cseE cells by a Quick-RNA Miniprep Plus kit (Zymo Research). The samples were treated with DNase (Roche Diagnostics) and RNA was purified with an RNA Clean&Concentrator-5 kit (Zymo Research). Ribosomal rRNA was removed with a Ribo-Zero rRNA Removal Kit for bacteria (Illumina). The purity of RNA was then tested with an Agilent RNA Pico 6000 kit and an Agilent 2100 Bioanalyzer (Agilent Technologies). TruSeq Stranded mRNA Sample Preparation guide (Illumina) was then used to construct the cDNA library. The constructed cDNA library was then sequenced with Illumina HiSeq 1500 using a read length of 74 bases.
2023-01-11 | E-MTAB-12457 | biostudies-arrayexpress
Project description:We performed RNA-sequencing experiments to examine gene expression in the genome of the cold-adapted (psychrophilic) marine diatom Fragilariopsis cylindrus to six different treatments simulating conditions found within sea ice. RNA-seq was performed on replicate samples for each experimental condition. Phytoplankton cells were grown under six different experimental conditions including (I) 0℃ and salinity 34 (seawater before ice formation), (II) two days at -4℃ and salinity 34 (early freezing or frazil ice formation), (III) eight days at -4℃ and salinity 34 (late freezing, frazil ice layer formation), (IV) two days at -4℃ and salinity 52 (initialization of brine channel formation in ice), (V) eight days at -4℃ and salinity 52 (established brine channel formation and young consolidated columnar ice), and (VI) two days at -8℃ and salinity 52 (further temperature stress). Total RNA was extracted using a guanidinium thiocyanate-phenol-chloroform extraction protocol, followed by DNase I treatment and RNA purification (Quiagen). First strand cDNA synthesis was performed using random hexamers. Library preparation was performed using the RNA-seq Sample Prep Kit (Illumina) and sequencing was conducted according to the TruSeq RNA sequencing protocol (Illumina). Illumina TruSeq RNA library production process: The libraries for this project were constructed with the PerkinElmer Sciclone using the TruSeq RNA protocol (Illumina 15026495 Rev.B). The library preparation involved an initial QC of the RNA using Qubit DNA (Life technologies Q32854) and RNA (Life technologies Q32852) assays as well as a quality check using the Bioanalyzer with the Nano kit (Agilent 5067-1511). 1ug of this RNA was purified to extract mRNA with a poly- A pull down using biotin beads and fragmented, first strand cDNA was synthesised followed by the second strand. The ends of the samples were repaired using the 3' to 5' exonuclease activity to remove the 3' overhangs and the polymerase activity to fill in the 5' overhangs creating blunt ends. A single ‘A’ nucleotide was added to the 3’ ends of the blunt fragments to prevent them from ligating to one another during the adapter ligation reaction. A corresponding single ‘T’ nucleotide on the 3’ end of the adapter provided a complementary overhang for ligating the adapter to the fragment. This strategy ensured a low rate of chimera formation. The ligation of a number indexing adapters to the ends of the DNA fragments prepared them for hybridisation onto a flow cell. The ligated products were subjected to a bead based size selection using Beckman Coulter XP beads (Beckman Coulter A63880). This removed the majority of un-ligated adapters, as well as any adapters that may have ligated to one another. Prior to hybridisation to the flow cell the samples were PCR’d to selectively enrich those DNA fragments that have adapter molecules on both ends and to amplify the amount of DNA in the library. The PCR was performed with a PCR primer cocktail that annealed to the ends of the adapter. The insert size of the libraries was verified by running an aliquot of the DNA library on a PerkinElmer GX using the High Sensitivity DNA chip (PerkinElmer CLS760672) and the concentration was determined by using a High Sensitivity Qubit assay and q-PCR. Illumina HiSeq 2000 clustering and sequencing: The 24 libraries were normalised and equimolar pooled to 10 nM using elution buffer (Qiagen). Each library pool was then diluted to 2 nM with NaOH and 5μL transferred into 995μL HT1 (Illumina) to give a final concentration of 10pM. 120 μL of each diluted library pool was then transferred into a 200 μL strip tube, spiked with 1% PhiX Control v3 and placed on ice before loading onto the Illumina cBot. The flow cell was clustered using TruSeq Paired End Cluster Generation Kit v3, following the Illumina PE_amplification_Linearization_Blocking_PrimerHyb recipe. Following the clustering procedure, the flow cell was loaded onto the Illumina HiSeq2000 instrument following the manufacturer’s instructions. The sequencing chemistry used was TruSeq SBS Kit v3-HS using HiSeq Control Software 1.4.8 and RTA 1.12.4.2. Each library pool was run in a single lane (lane 5 and 6) for 50cycles of each paired end read. Reads in bcl format were demultiplexed based on the 6 bp Illumina index by CASSAVA, allowing for a one base-pair mismatch per library, and converted to FASTQ format by bcl2fastq.
2018-01-18 | E-MTAB-5153 | biostudies-arrayexpress
Project description:To detect Transcription Start sites (TSS) in Staphylococcus aureus wildtype a cDNA library enriched for primary 5′-transcripts according to Peifer-Sancar et al. (2013) was prepared and sequenced on Illumina MiSeq system to retrieve 5′-ends of primary transcripts. Cultivation was performed in Luria Bertani (LB) medium in triplicate at 37°C until cells have reached an optical density at 540 nm of 2.0. Cells were harvested by centrifugation, washed with Belitsky minimal medium (BMM) and adapted to BMM for one hour. S. aureus cells of 3 replicate experiments were harvested and disrupted in 3 mM EDTA/ 200 mM NaCl lysis buffer with a Precellys24 Ribolyzer. RNA isolation was performed using the phenol-chloroform-isoamylalcohol approach. After precipitation with 3 M sodium acetate and isopropanol, the total RNA was washed with cold ethanol and the pellet was solved in sterile water. Initially RNA quality was checked by Trinean Xpose (Gentbrugge,Belgium) and Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). Samples contaminated with DNA were treated with DNase (Qiagen), cleaned as described above and rechecked by Xpose and Agilent Bioanalyzer. Finally RNA was free of DNA with an RNA Integrity Number (RIN) > 9 and rRNA Ratio [23s / 16s] > 1.5. Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the ribosomal RNA molecules from the isolated total RNA. Removal of rRNA was checked by Agilent RNA Pico 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). RNA was free of detectable rRNA. The method from Peifer-Sancar et al. (2013) was used to prepare a cDNA library enriched for primary 5′-transcript. The resulting cDNAs were sequenced paired end on an Illumina MiSeq system (San Diego, CA, USA) using 75 bp read length.
2019-04-01 | E-MTAB-7385 | biostudies-arrayexpress
Project description:To detect Transcription Start sites (TSS) in Staphylococcus aureus a cDNA library enriched for primary 5′-transcripts according to Peifer-Sancar et al. (2013) was prepared and sequenced on Illumina MiSeq system to retrieve 5′-ends of primary transcripts. Cultivation was performed in Luria Bertani (LB) medium in triplicate at 37°C until cells have reached an optical density at 540 nm of 2.0. Cells were harvested by centrifugation, washed with Belitsky minimal medium (BMM) and adapted to BMM for one hour. S. aureus cells of 3 replicate experiments were harvested before and 30 min after exposure to 300 µM allicin and disrupted in 3 mM EDTA/ 200 mM NaCl lysis buffer with a Precellys24 Ribolyzer. RNA isolation was performed using the phenol-chloroform-isoamylalcohol approach. After precipitation with 3 M sodium acetate and isopropanol, the total RNA was washed with cold ethanol and the pellet was solved in sterile water. Initially RNA quality was checked by Trinean Xpose (Gentbrugge,Belgium) and Agilent RNA Nano 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). Samples contaminated with DNA were treated with DNase (Qiagen), cleaned as described above and rechecked by Xpose and Agilent Bioanalyzer. Finally RNA was free of DNA with an RNA Integrity Number (RIN) > 9 and rRNA Ratio [23s / 16s] > 1.5. Ribo-Zero rRNA Removal Kit (Bacteria) from Illumina (San Diego, CA, USA) was used to remove the ribosomal RNA molecules from the isolated total RNA. Removal of rRNA was checked by Agilent RNA Pico 6000 kit on Agilent 2100 Bioanalyzer (Agilent Technologies, Böblingen, Germany). RNA was free of detectable rRNA. The method from Peifer-Sancar et al. (2013) was used to prepare a cDNA library enriched for primary 5′-transcript. The resulting cDNAs were sequenced paired end on an Illumina MiSeq system (San Diego, CA, USA) using 75 bp read length.
2021-10-25 | E-MTAB-10889 | biostudies-arrayexpress
Project description:Most of small RNA library construction methods are based on RNA ligases, which prefer to join the molecules (small RNAs and adapters) that can anneal to each other and form a ligase favoured structure. Different platforms for next generation sequencing use different adapter sequences, causing the cloning bias. Adapters with degenerated nucleotides at the ligating ends (High Definition, HD adapters) were developed to reduce the cloning bias. However, above 90% of the cloning products is adapter dimer when the current available commercial kits and their corresponding protocols are used. Here we adopted and further improved a method demonstrated in a publically available patent (http://www.google.com/patents/WO2011056866A2?cl=en). Using the improved method, we constructed the small RNA libraries by using the total RNA of Medicago truncatula leaf tissue. The adapter dimer was significantly reduced. The small RNA sequences were also analysed. The small RNA libraries of medicago truncatula leaves were constructed by using an improved protocol where high-definition (HD) adapters were used.
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2015-01-31 | E-GEOD-59331 | biostudies-arrayexpress
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2014-04-15 | E-ERAD-228 | biostudies-arrayexpress
Project description:Total RNA was extracted from morpholigcally abnormal and wildtype sibling larvae of neural crest mutants derived from ENU mutagenesis. The 3' end of fragmented RNA was pulled down using polyT oligos attached to magnetic beads, reverse transcribed, made into Illumina libraries and sequenced using IlluminaHiSeq paired-end sequencing. Protocol: Total RNA obtained was DNase treated. Chemically fragmented RNA was enriched for the 3' ends by pull down using an anchored polyToligo attached to magnetic beads. An RNA oligo comprising part of the Illumina adapter 2 was ligated to the 5' end of the captured RNA and the RNA was eluted from the beads. Reverse transcription was primed with an anchored polyToligo with part of Illumina adapter 1 at the 5' end followed by 4 random bases, then an A, C or G base, the one of twelve 5 base indexing tags and 14T bases. An Illumina library with full adapter sequence was produced by 15 cycles of PCR. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
2014-01-07 | E-ERAD-203 | biostudies-arrayexpress
Project description:Total RNA was extracted from morpholically abnormal and sibling wild type embryos identified by the Zebrafish Mutation Project (http://www.sanger.ac.uk/Projects/D_rerio/zmp/). The 3 end of fragmented RNA was pulled down using polyToligos attached to magnetic beads, reverse transcribed, made into Illumina libraries and sequenced using Illumina HiSeq paired-end sequencing. Protocol: Total RNA was extracted from zebrafish embryos using Trizol and DNase treated. Chemically fragmented RNA was enriched for the 3 ends by pulled down using an anchored polyToligo attached to magnetic beads. An RNA oligo comprising part of the Illumina adapter 2 was ligated to the 5 end of the captured RNA and the RNA was eluted from the beads. Reverse transcription was primed with an anchored polyT oligo with part of Illumina adapter 1 at the 5 end followed by 12 random bases, then an 8 base indexing tags, then CG and 14 T bases. An Illumina library with full adapter sequence was produced by 20 cycles of PCR. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
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Project description:Total RNA was extracted from morpholically abnormal and sibling wild type embryos identified by the Zebrafish Mutation Project (http://www.sanger.ac.uk/Projects/D_rerio/zmp/). The 3 end of fragmented RNA was pulled down using polyToligos attached to magnetic beads, reverse transcribed, made into Illumina libraries and sequenced using IlluminaHiSeq paired-end sequencing. Protocol: Total RNA was extracted from mouse embryos using Trizol and DNase treated. Chemically fragmented RNA was enriched for the 3 ends by pulled down using an anchored polyToligo attached to magnetic beads. An RNA oligo comprising part of the Illumina adapter 2 was ligated to the 5 end of the captured RNA and the RNA was eluted from the beads. Reverse transcription was primed with an anchored polyToligo with part of Illumina adapter 1 at the 5 end followed by 4 random bases, then an A, C or G base, then one of twelve5 base indexing tags and 14 T bases. An Illumina library with full adapter sequence was produced by 15 cycles of PCR. This data is part of a pre-publication release. For information on the proper use of pre-publication data shared by the Wellcome Trust Sanger Institute (including details of any publication moratoria), please see http://www.sanger.ac.uk/datasharing/
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