Samples of HOCl-treated and untreated CFT073 and ΔrcrR cells were collected as described before for qRT-PCR. After extraction of total RNA (Macherey & Nagel) and removal of the residual DNA using the TURBO DNA-free kit (Thermo Scientific), rRNA was depleted using the Illumina Ribo Zero Kit (Illumina) for Gram-negative bacteria. A total of 150 bp single-end sequencing was performed on an Illumina HiSeq 2500 by Novogene (Sacramento, USA). Differential gene expression analysis of three biological replicates, including normalization, was performed in the bioinformatics platform Galaxy (101 (link)). Briefly, RNAseq reads were mapped to E. coli CFT073 reference sequence (GCA_000007445.1 ) using HISAT2 (102 (link)). Then, the number of reads mapped to each gene were counted using featureCounts (103 (link)). Finally, differential gene expression was determined using DESeq2 (104 (link)) with an adjusted P value cut off P ≤ 0.05 and logFC cut off 1.5.
Ribo zero kit
Manufactured by Illumina
Sourced in United States, Singapore, Germany
The Ribo-Zero kit is a laboratory equipment product designed for the depletion of ribosomal RNA (rRNA) from total RNA samples. The kit utilizes a hybridization-based approach to selectively remove rRNA molecules, allowing for the enrichment of other RNA species, such as messenger RNA (mRNA), for downstream analysis.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500, by Illumina (31 mentions)
Trizol, by Thermo Fisher Scientific (18 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (16 mentions)
Rneasy mini kit, by Qiagen (15 mentions)
Hiseq 2000, by Illumina (14 mentions)
Trizol reagent, by Thermo Fisher Scientific (14 mentions)
Nebnext ultra rna library prep kit for, by Illumina (10 mentions)
Rneasy kit, by Qiagen (9 mentions)
Qubit 2, by Thermo Fisher Scientific (9 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (8 mentions)
Agilent 2200 tapestation, by Thermo Fisher Scientific (8 mentions)
Turbo dnase, by Thermo Fisher Scientific (7 mentions)
Hiseq 4000, by Illumina (6 mentions)
Illuminahiseq 3000 platform, by Illumina (6 mentions)
Nextseq 500, by Illumina (6 mentions)
Hiseq x ten platform, by Illumina (5 mentions)
Scriptseq 2.0 kit, by Illumina (5 mentions)
Nebnext ultra directional rna library prep kit for, by Illumina (5 mentions)
Novaseq 6000, by Illumina (5 mentions)
Bioanalyzer, by Agilent Technologies (5 mentions)
215 protocols using ribo zero kit
1
Differential Gene Expression Analysis of E. coli CFT073
2
RNA-seq analysis of E. coli cisplatin response
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Four biological replicates of wild-type E. coli MG1655 and Δppk mutant were cultivated in MOPS-G medium at 37°C until an OD600 of 0.4 to 0.5 was reached. Cells (1 ml) were harvested either before or 15 min after the treatment with 20 μg/ml cisplatin in 1 ml of ice-cold methanol (−80°C) to stop transcription. After centrifugation and removal of the supernatant, total RNA was prepared using the Ambion RiboPure-Bacteria kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The samples were DNase I treated, followed by depletion of rRNA using the Illumina Ribo Zero kit (Illumina) for Gram-negative bacteria. Fifty-base single-end sequencing was performed on an Illumina HiSeq 4000 using the University of Michigan DNA Sequencing Core. Sequence reads from the RNA-seq were mapped onto the reference genome (NC_000913 ). Genes with a log2 fold change of >0.5 and a false-discovery rate (FDR) value of <0.01 were considered differentially expressed genes (DEGs).
3
Transcriptomic Analysis of Bacteria Using RNA-Seq
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from exponentially growing host-independent B. bacteriovorus HID13 and from −80 °C-frozen Aquifex aeolicus pellets (purchased from the Archaeenzentrum Regensburg, Germany) using the RNEasy kit (Qiagen), including DNase I treatment. RNA quality was assessed using an Agilent 2100 Bioanalyser.
For RNA extraction from B. bacteriovorus HID13, cells were diluted in 10 ml of fresh YP medium at an optical density (OD) of 0.1 and grown until reaching a density of 0.6 (20 h at 30 °C with shaking). For each replicate, 2 ml of culture were spun at maximum speed, snap frozen and kept at −80 °C until RNA extraction.
For the B. bacteriovorus samples (all RNA integrity number (RIN) scores >9.5), ribosomal RNA was depleted using the NEBNext rRNA depletion kit (Bacteria) and libraries made using the NEBNext Ultra II Directional RNA Library prep kit for Illumina according to manufacturer instructions. Paired-end 55 bp reads were generated on a NextSeq 2000 sequencing system with dual 8 bp indexing. For the A. aeolicus samples (all RIN scores >5), rRNA was depleted using an Illumina Ribozero kit (Bacteria) and libraries made using the TruSeq Stranded Total RNA LT kit according to manufacturer instructions. Single-end 50 bp reads were generated on a MiSeq with single 6 bp indexing.
For RNA extraction from B. bacteriovorus HID13, cells were diluted in 10 ml of fresh YP medium at an optical density (OD) of 0.1 and grown until reaching a density of 0.6 (20 h at 30 °C with shaking). For each replicate, 2 ml of culture were spun at maximum speed, snap frozen and kept at −80 °C until RNA extraction.
For the B. bacteriovorus samples (all RNA integrity number (RIN) scores >9.5), ribosomal RNA was depleted using the NEBNext rRNA depletion kit (Bacteria) and libraries made using the NEBNext Ultra II Directional RNA Library prep kit for Illumina according to manufacturer instructions. Paired-end 55 bp reads were generated on a NextSeq 2000 sequencing system with dual 8 bp indexing. For the A. aeolicus samples (all RIN scores >5), rRNA was depleted using an Illumina Ribozero kit (Bacteria) and libraries made using the TruSeq Stranded Total RNA LT kit according to manufacturer instructions. Single-end 50 bp reads were generated on a MiSeq with single 6 bp indexing.
4
RNA-seq Analysis of Human Transcriptome
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Isolated total RNA was subjected to ribosomal RNA depletion using a RiboZero Kit (Epicentre, Madison, WI, USA), cut randomly into short fragments and then reverse-transcribed into cDNA (Illumina, San Diego, CA, USA). Sequencing adapters were ligated to both ends of the cDNA fragments. After amplification of the adapter-ligated fragment by PCR, fragments with inserts size between 200 and 400 base pairs (bp) were selected and sequenced using an Illumina HiSeqTM 2500 sequencer (Illumina) according to the standard Illumina protocol. Analysis of the quality and filtering of the raw RNA-sequencing (seq) data to remove low quality reads, adaptor sequences, contaminant DNA and PCR duplicates was performed using the Trimmomatic software version 0.32 (Illumina). The trimmed RNA-seq reads were mapped to the human reference genome hg 19 using TopHat2 software version 2.0.13.10 Cufflinks software version 1.0.3 was used to process the reads and calculate the transcription level of each gene (University of Washington, Seattle, WA, USA). To obtain the differential expression profiles, the fragment per kilobase of transcript per million mapped reads (FPKM) values were calculated for each lncRNA.
5
Neuronal RNA-seq Transcriptomics Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was extracted from neurons using RNeasy mini Kit (Qiagen, Cat# 74104). Ribosomal RNA was depleted with RiboZero kit (Epicentre, Cat# MRZH116). Poly(A)-enriched RNA were separated by three rounds of Oligo(dT) magnetic beads (Thermo, Cat# 61002). For each time point, RNA from two 60-mm dishes of neurons were pooled together. Sequence quality was assessed using FastQC, and quality trimming was done using Trimmomatic.69 (link) RNA-seq reads were aligned to the rat genome (iGenomes UCSC version rn6) using TopHat v2.0.9, and only uniquely mapped reads with at most two mismatches were considered for downstream analysis. Gene count tables were constructed using HTseq70 (link) and used as input for edgeR 3.0.8 71 (link). The list of activity regulated genes is generated based on previous in vivo studies.75 (link)–77 (link)
6
Strand-specific mRNA-seq library preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
rRNA was removed using the Ribo-Zero Kit (Epicentre). rRNA-depleted RNA was fragmented with an average length of 100 to 200 base pairs (bp) and converted to double-stranded complementary DNA (cDNA). Library preparation was done using a protocol based on the “dUTP (deoxyuridine triphosphate) method,” to generate strand-specific mRNA-seq libraries including barcoding (38 (link), 39 (link)). The Illumina stranded TruSeq RNA-seq library preparation kit was used. Sequencing of the library was done using the Illumina HiSeq: single-end reads, one lane, 50 cycles, two samples per lane. The sequencing data produced were processed, removing low-quality sequence reads. Furthermore, the sequence data in FastQ format were additionally filtered and trimmed on the basis of Phred quality scores.
7
Illumina-based Transcriptome Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was digested with DNaseI and depletion of ribosomal RNA was performed using the Ribo-Zero kit (Epicentre) for Gram-negative bacteria. Integrity of the prepared RNA was confirmed using an Agilent 2100 Bioanalyzer. Directional cDNA libraries were prepared using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB E7760) according to the manufacturer's instructions and cDNA library quality was tested on an Agilent 2100 Bioanalyzer. The libraries were sequenced using a HiSeq 1500 machine (Illumina) in single-read mode with 100 bp read length. The sequencing data has been deposited at Gene Expression Omnibus (GEO) under the GSE115711 accession code.
8
Transcriptome Sequencing of CNE-2 and CNE-2-Rs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted from CNE-2 and CNE-2-Rs cells, and the Ribo-Zero™ Kit (Epicentre, Madison, WI, USA) was used to remove the rRNA. The remaining RNA was cut randomly into short fragments. First-strand cDNA was transcribed based on these random fragments using random hexamers; second-strand cDNA was transcribed by mixing the first-strand cDNA with buffer, dNTPs, RNase H, and DNA polymerase I. Short fragments were purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) and resolved with EB buffer for end reparation and single nucleotide A (adenine) addition. The short fragments were connected with adapters and the second strand was degraded using UNG (uracil-N-glycosylase). RNA fragments were separated by agarose gel electrophoresis, and the fragments were expanded with polymerase chain reaction (PCR). The PCR products were sequenced using an Illumina HiSeq™ 2000 instrument (Illumina Inc., San Diego, CA, USA), and the original image data were converted into “.fq” files by base calling software. The relative data were submitted to NCBI under BioProject accession No. PRJNA 254709. The details of the experiment were as follows: expected library size: 200 bp; read length: 90 nt; and sequencing strategy: paired-end sequencing.
9
Comprehensive RNA Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
1 µg of total RNA was treated with Turbo-DNase (Life Technologies) according to the manufacturer’s instructions. Then, rRNA was removed from samples using a Ribo-Zero Kit (Epicentre), according to the manufacturer’s protocol, and samples were spiked-in with external RNA (ERCC RNA Spike-In Mix; Life Technologies). For libraries prepared from poly(A)+ fractions, the ribo-depletion step was omitted. Finally, RNA libraries were prepared using a TruSeq RNA Sample Preparation Kit (Illumina) or a KAPA Stranded RNA-Seq Library Preparation Kit (Kapa Biosystems) according to the manufacturer’s instructions. Quality of libraries was determined by chip electrophoresis performed using the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.).
10
Transcriptome Profiling of Coelomocytes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNAs were extracted from coelomocytes using Trizol (Thermo Fisher, USA) according to the manufacturer’s protocol. The ribosomal RNA was removed using the Ribo-Zero™ kit (Epicentre, USA). Fragmented RNAs were subjected to the first-strand and second strand cDNA synthesis, followed by adaptor ligation and enrichment with a low-cycle according to instructions of NEBNext® Ultra™ Directional RNA Library Prep Kit for Illumina (New England Biolabs Incorporation, USA). The purified library products were evaluated by Agilent 2100 Bioanalyzer (Life Technologies, USA). The libraries were paired-end sequenced by Cloudseq Biotech Inc. (Shanghai, China) using IlluminaHiSeq 4000 platform.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!