Overnight cultures of T6SS+, T6SS−, and WT Vibrio cholerae were grown at 37°C in lysogeny broth (LB) (n = 2 biological replicates). RNA was extracted from these cultures using an Qiagen RNeasy mini kit (Qiagen, Hilden, Germany). An RNA library was prepared using the NEB Ultra II directional RNA library prep kit (New England Biolabs, Ipswitch, MA, USA) and the QIASeq Fast select ‐RNA HMR kit (Qiagen, Hilden, Germany) was used for bacterial rRNA depletion. The completed bacterial RNA library was sequenced on the Illumina NovaSeq 6000 (Illumina, San Diego, CA, USA). To analyze the transcriptomic data, we first trimmed adapters and filtered low‐quality reads using Trimmonatic (v0.39) (Bolger et al., 2014 (link)). We then perform alignment of paired reads to reference contigs of V. cholerae C6706 (NCBI RefSeq assembly GCF_009763945.1) using STAR (v 2.7.11a) (Dobin et al., 2013 (link)) to create binary alignment files (BAM) sorted by genomic coordinates. We counted aligned fragments to all annotated loci in NCBI Refseq annotation using featureCounts (v2.0.6) (Liao et al., 2014 (link)). Fragment counts were filtered for low expression and used for differential expression analysis using DESeq2 in R (Love et al., 2014 (link)).
Neb ultra 2 directional rna library prep kit
Manufactured by New England Biolabs
Sourced in United States
The NEB Ultra II Directional RNA Library Prep Kit is a laboratory equipment product designed for the preparation of directional RNA libraries. The kit provides the necessary reagents and protocols for the creation of sequencing-ready libraries from RNA samples.
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Lab products found in correlation
Hiseq 2500 platform, by Illumina (2 mentions)
Nanodrop 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Novaseq s2 pe100 bp lane, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Protein g beads, by Thermo Fisher Scientific (1 mentions)
Isopropanol, by Thermo Fisher Scientific (1 mentions)
Chloroform, by Thermo Fisher Scientific (1 mentions)
Depc treated water, by Thermo Fisher Scientific (1 mentions)
Tri reagent, by Merck Group (1 mentions)
Rnaeasy plus micro kit, by Qiagen (1 mentions)
Ribominus eukaryote kit, by Thermo Fisher Scientific (1 mentions)
6 protocols using neb ultra 2 directional rna library prep kit
1
Transcriptional Profiling of T6SS Vibrio cholerae
2
VNPP433-3β Transcriptome Analysis in CWR22Rv1 Cells
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Total RNA was isolated from CWR22Rv1 cells treated with 10 μM VNPP433‐3β (24 h) using RNeasy Plus mini kit (Qiagen) following manufacturer's instructions. DMSO vehicle control and drug treatment were performed in triplicates and all RNA samples were quantified and quality‐checked using Agilent 2100 Bioanalyzer. Only RNA preps having an RNA Integrity Number (RIN) of 8 or above were considered for RNA‐seq experiments. NEB Ultra II Directional RNA library prep kit was used for preparing RNA‐Seq libraries. The libraries thus generated were evaluated for size distribution and yield using Qubit and Agilent 2100 Bioanalyzer. RNA sequencing was conducted on Illumina NovaSeq S2 PE100 bp lane at Maryland Genomics, Institute for Genome Sciences, University of Maryland, Baltimore. The quality of sequencing was measured by Phred quality score (Q score) and more than 90% of the sequencing reads attained Q30 (99.9% base call accuracy). GO and GSEA were carried out using GSEA v4.2.2 against Molecular Signatures Database v7.5.1 with permutation set at “gene set” and other parameters default.15 Qiagen IPA was carried out to uncover canonical pathways modulated by VNPP433‐3β.
3
m6A-Seq Protocol for Mouse Tissue
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m6A‐seq and library preparation were conducted according to a previously published protocol with following modifications.4 Briefly, 100 μg total RNAs were extracted from left ventricular tissues of 8‐week‐old male mice using TRIzol following the manufacturer's protocol. Fragmented mRNAs were immunoprecipitated with anti‐m6A antibody (56593, CST) in IP buffer (150 mM NaCl, 0.1% NP‐40, and 10 mM Tris‐HCl, pH 7.4) for 3 h, at 4 °C, and 1/10 of the fragmented mRNA was saved as input. The antibody‐RNA complex was isolated by incubation with protein G beads (10004D, Thermo Fisher) for 2 h, at 4 °C. The beads were washed three times and eluted competitively with m6A monophosphate solution. The RNA‐seq libraries were prepared using a NEB Ultra II Directional RNA Library Prep Kit (E7760, NEB).
4
RNA Extraction from Zebrafish Brain Tissue
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RNA was prepared from brain tissue of 12-week-old zebrafish. A TRIzol-based protocol was used to extract RNA from the tissue. Briefly, individual brains were homogenised in 250 µl TRI Reagent (Sigma-Aldrich) and incubated at room temperature before adding 50 µl chloroform (Thermo Fisher Scientific). The samples were centrifuged at 13,300 g and the top aqueous phase was collected and transferred to a separate tube. RNA was precipitated from the aqueous phase by mixing with an equal volume of isopropanol (Thermo Fisher Scientific) and centrifugation at 13,300 g . The precipitated RNA was resuspended in DEPC-treated water (Thermo Fisher Scientific), and its concentration and quality were quantified using the Nanodrop 1000 Spectrophotometer. Approximately 750 ng of high-quality total RNA, with an RNA integrity number of 9 or above, was used in the preparation of sequencing libraries using the NEB Ultra II Directional RNA Library Prep Kit (New England Biolabs, E7760), following the polyA mRNA workflow [NEBNext® Poly(A) mRNA Magnetic Isolation Module]. Libraries were individually indexed and pooled for sequencing. Single-end 100 bp sequencing was performed on the Illumina HiSeq 2500 platform using Rapid Run mode with V2 chemistry.
5
Single-cell RNA-seq of Epithelial Cells
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Two biological replicates (n = 2) at E13.5 were used for bulk RNA-seq experiments. ShhCre/+;R26mTmG/+ mice were used to isolate lung epithelial cells, while Pdx1Cre/+;R26mTmG/+ mice were used to isolate pancreatic epithelial cells. Tissues were prepared for sorting in the same manner as E9.5 samples but did not undergo EPCAM staining. Live, GFP-expressing cells were sorted and used for RNA isolation using the RNAeasy Micro Plus kit (Qiagen, 74034). RNA libraries were prepared using the NEB Ultra II Directional RNA Library Prep Kit (New England Biolabs, E7765). SIRV-Set 3 synthetic RNA spike-in was used at the manufacturer’s recommendations to help normalize batch effects (Lexogen, 051.01) Samples underwent paired-end sequencing (2 × 125 base pair) on the Illumina HiSeq 2500 platform.
6
RNA-seq library construction from mouse hearts
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Total RNAs from left ventricular tissues of 2‐week‐old and 8‐week‐old male mice were extracted to construct RNA‐seq libraries. RNA samples were pre‐treated with RiboMinus Eukaryote Kit (A15020, Thermo Fisher) to remove ribosomal RNAs. The remaining intact RNA was fragmented, and RNA‐seq libraries were prepared by using a NEB Ultra II Directional RNA Library Prep Kit (E7760, NEB). End‐repaired fragments were ligated with a unique Illumina adapter.
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