RNA-sequencing libraries were prepared from mid log cells grown in LB with the NEB Ultra II Directional RNA Library Prep Kit for Illumina (NEB, E7760S). Reads were trimmed with Cutadapt and then aligned to the E. coli genome with Bowtie2 (ref. 62 (link)). DESeq2 (ref. 65 (link)) was used to analyse differential expression.
Neb ultra 2 directional rna library prep kit
Manufactured by Illumina
The NEB Ultra II Directional RNA Library Prep Kit is a laboratory equipment used for the preparation of directional RNA libraries for sequencing. It provides a streamlined workflow for generating high-quality libraries from total RNA samples.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 4000, by Illumina (1 mentions)
Rnaeasy micro kit, by Qiagen (1 mentions)
Ipa software, by Qiagen (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Rna 6000 nano kit, by Agilent Technologies (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Nextseq 2000 platform, by Illumina (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Nebnext multiplex oligo, by Illumina (1 mentions)
4 protocols using neb ultra 2 directional rna library prep kit
1
RNA-seq Analysis of E. coli Transcriptome
2
RNA-Seq of Hematopoietic Stem and Progenitor Cells
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RNA from HSCs (LSK CD150+CD48–) and CLPs (LIN– SCA-1loC-KITloCD127+) from control and Zeb1–/– mice 14 days after the last dose of pIpC injection was extracted using the RNAeasy Micro Kit (QIAGEN). Total RNA quality and quantity were assessed using Agilent 2100 Bioanalyzer and the RNA Nano 6000 Kit (Agilent Technologies). The library was prepared using the NEB Ultra II Directional RNA Library Prep Kit for Illumina. The libraries then were sequenced using a 75 base paired end (2 × 75bp PE) dual index read format on the Illumina HiSeq4000 according to the manufacturer’s instructions. Further details on sequencing and bioinformatics were described previously (66 (link)).
The heatmap was created using Morpheus, an online tool, (Broad Institute). DEGs with an FDR of less than 0.05 were used for heatmaps. The biological pathway analysis was performed using BioCarta, KEGG, and Reactome pathway databases run on GSEA software (94 (link)) as well as IPA software (QIAGEN). IPA was used to create a prediction network of Zeb1 interactions with its target genes.
RNA-Seq for EPCAM+ and EPCAM– from Zeb1–/– mice is described inSupplemental Methods .
All RNA-Seq data are available in the NCBI’s GEO database (GSE153664, GSE154615).
The heatmap was created using Morpheus, an online tool, (Broad Institute). DEGs with an FDR of less than 0.05 were used for heatmaps. The biological pathway analysis was performed using BioCarta, KEGG, and Reactome pathway databases run on GSEA software (94 (link)) as well as IPA software (QIAGEN). IPA was used to create a prediction network of Zeb1 interactions with its target genes.
RNA-Seq for EPCAM+ and EPCAM– from Zeb1–/– mice is described in
All RNA-Seq data are available in the NCBI’s GEO database (GSE153664, GSE154615).
3
RNA-seq Analysis of C. glabrata Mutant
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2 h and 10 h RPMI-medium and macrophage-internalized wt and Cgsnf2Δ cells were harvested and total RNA was extracted using RNeasy kit (Qiagen), followed by removal of DNA contamination, if any, using DNase I. The purified total RNA was sent to National Genomics Core (NGC) at CDFD, Hyderabad (http://ngc.cdfd.org.in/ ) for sequencing, which involved mRNA enrichment using Poly(A) mRNA Magnetic Isolation Module, library preparation using NEB Ultra II Directional RNA Library Prep Kit and 150 bp paired-end sequencing on the Illumina NextSeq 2000 platform. Sequenced reads were processed and mapped on to the C. glabrata CBS138 reference genome using HISAT 2.1.0 aligner.
For gene expression quantification, the counts of mapped reads for each gene were considered using the Feature counts tool, followed by sequencing depth normalization of raw read counts using the DESeq2 tool. Differentially expressed genes were classified based on two criteria: ≥ 2-fold change in expression and adjusted p value of ≤0.05.
For gene expression quantification, the counts of mapped reads for each gene were considered using the Feature counts tool, followed by sequencing depth normalization of raw read counts using the DESeq2 tool. Differentially expressed genes were classified based on two criteria: ≥ 2-fold change in expression and adjusted p value of ≤0.05.
4
RNA-seq Library Preparation and Sequencing
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RNA was extracted using the QIAGEN RNeasy Mini kit with DNase I treatment. RNA integrity numbers were confirmed using a Tapestation RNA screentape to be above 9.8, and a Qubit High Sensitivity assay was used to determine RNA concentration.
Ribosomal RNA was removed using the NEB rRNA Depletion Kit (Human/Mouse/Rat) using 500 ng of RNA as input. Following depletion, RNA-seq libraries were prepared using the NEB Ultra II Directional RNA Library Prep Kit for Illumina, and NEBNext Multiplex Oligos for Illumina. Library concentration and fragment size was determined using Qubit (dsDNA HS assay) and Tapestation (D1000 screentape). Libraries from each timepoint were pooled to a final DNA concentration of 15 nM, and 75-bp paired-end reads were sequenced on an Illumina NextSeq 500 using a High Output Kit.
Ribosomal RNA was removed using the NEB rRNA Depletion Kit (Human/Mouse/Rat) using 500 ng of RNA as input. Following depletion, RNA-seq libraries were prepared using the NEB Ultra II Directional RNA Library Prep Kit for Illumina, and NEBNext Multiplex Oligos for Illumina. Library concentration and fragment size was determined using Qubit (dsDNA HS assay) and Tapestation (D1000 screentape). Libraries from each timepoint were pooled to a final DNA concentration of 15 nM, and 75-bp paired-end reads were sequenced on an Illumina NextSeq 500 using a High Output Kit.
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