mRNA was isolated from 1 μg DNAse-treated total RNA using the Ribo-Zero Gold Kit (human, mouse, rat) from Illumina according to the manufacturer's instructions. Final elution was done in 5 μl nuclease free water. Samples were then directly subjected to the workflow for strand-specific RNA-Seq library preparation (Ultra Directional RNA Library Prep II, NEB). For ligation, custom adaptors were used 1: (Adaptor-Oligo 5′-ACA CTC TTT CCC TAC ACG ACG CTC TTC CGA TCT-3′, Adaptor-Oligo 2: 5′-P-GAT CGG AAG AGC ACA CGT CTG AAC TCC AGT CAC-3′). After ligation, adapters were depleted by an XP bead purification (Beckman Coulter) adding beads in a ratio of 1:1. Indexing was done during the following PCR enrichment (15 cycles) using custom amplification primers carrying the index sequence indicated with ‘NNNNNN’. (Primer1: Oligo_Seq AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC T, primer2: GTG ACT GGA GTT CAG ACG TGT GCT CTT CCG ATC T, primer3: CAA GCA GAA GAC GGC ATA CGA GAT NNNNNN GTG ACT GGA GTT. After two more XP beads purifications (1:1) libraries were quantified using Qubit dsDNA HS Assay Kit (Invitrogen). Samples were equimolarly pooled and used for 75bp single read sequencing on a Nextseq 500 (Illumina).
Ultra directional rna library prep 2
Manufactured by New England Biolabs
Sourced in United States
The Ultra Directional RNA Library Prep II is a library preparation kit designed for the generation of directional RNA-sequencing libraries. It enables the preparation of high-quality libraries from low input RNA samples while maintaining strand information.
Automatically generated - may contain errors
Lab products found in correlation
Nextseq 500, by Illumina (4 mentions)
Xp bead purification, by Beckman Coulter (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Ribo zero gold kit, by Illumina (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nucleospin rna xs, by Macherey-Nagel (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Nextseq, by Illumina (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Rneasy mini, by Qiagen (1 mentions)
6 protocols using ultra directional rna library prep 2
1
Strand-specific RNA-Seq Library Preparation
2
Transcriptome Analysis of Preadipocytes and Adipocytes
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Transcriptome analysis was performed as previously described [3 (link),65 (link)]. Briefly, aliquots of preadipocytes and adipocytes were used for RNA extraction, and mRNA was isolated from 1 µg RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module (BioLabs, Heidelberg, Germany). After fragmentation, the samples were subjected to the workflow for strand-specific RNAseq library preparation (Ultra Directional RNA Library Prep II, NEB) and 75 bp single-read sequencing was performed on an Illumina NextSeq500 (Shirley, NY, USA). After sequencing, FastQC was used to perform quality control. Differential expression between preadipocytes and adipocytes was tested with the R package DESeq2 v2.7.R [65 (link),68 (link)]. Selected gene expression data have been published previously [3 (link),65 (link)]. The complete data set is available under GEO accession GSE169514. A fold-change of ≥2 and ≤−2 and Benjamini–Hoch-berg-adjusted p values of ≤0.05 were used as criteria to select differentially expressed genes (DEG).
3
RNA-Seq Analysis of Human Islets and Pseudoislets
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Aliquots of cultured human islets and their respective pseudoislets were used for RNA extraction using NucleoSpin® RNA XS (Macherey-Nagel, Düren, Germany). RNA integrity (RIN ≥ 9) was measured with Bioanalyzer 2100 (Agilent Technologies). Sequencing was performed as described previously at the Center for Molecular and Cellular Bioengineering (Andreas Dahl, CMCB, Technical University Dresden, Germany)45 (link). In short, mRNA was isolated from 120 ng RNA by poly-dT enrichment using the NEBNext Poly(A) mRNA Magnetic Isolation Module. After fragmentation, the samples were subjected to the workflow for strand-specific RNASeq library preparation (Ultra Directional RNA Library Prep II, NEB) and 75 bp single read sequencing was performed on Illumina NextSeq500. After sequencing, FastQC was used to perform quality control. Differential expression between islets and pseudoislets was tested with DESeq R package (v.1.10.1). The complete analysis is available in Supplementary File 2 and under GEO code GSE133903.
4
RNA-Seq Analysis of AML Subtypes
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RNA sequencing was performed on total RNA isolated at diagnosis from 7 AML samples from UBTF-TDpos patients and 42 samples from other well established AML subgroups (t(8;21), t(6;9); inv16, NPM1 and in-frame CEBPAbZIP mutant patients, and patients with a NUP98-NSD1 fusion) using strand-specific RNA-Seq library preparation (Ultra II Directional RNA Library Prep, NEB) and sequenced on an Illumina NovaSeq 6000 instrument. The complete workflow as well as the bioinformatic analysis are detailed in the supplement.
5
Bacterial rRNA Depletion and Directional RNA Sequencing
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Bacterial rRNA was depleted using NEBNext Bacterial rRNA Depletion Kit (Catalog no. E7850X). The indexed cDNA library was generated using NEBNext Ultra II Directional RNA Library Prep (Catalog no. E7765L) and NEBNext Multiplex Oligos for Illumina (Catalog no. E6609S). In total, 40 samples (two biological replicates, 10 time points, two conditions) were prepped and sequenced. The library was sequenced at the Genetics Core in the Biological Nanostructures Laboratory at the University of California, Santa Barbara on an Illumina NextSeq with High Output, 150 Cycle, paired end settings.
6
Ankrd31 Deficiency in Mouse Testicular Transcriptome
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In order to test the effect of Ankrd31 deficiency in mouse testicular transcriptome total RNAs from Ankrd31 +/+ and Ankrd31 À/À juvenile testes (12 days old) were extracted. RNA was extracted using RNeasy Mini (QIAGEN, Cat No./ID: 74104) according to manufacturer's instructions. Quantification and quality control of RNA was performed using Agilent 2100 Bioanalyzer. mRNA was isolated from 300 ng total RNA by poly-dT enrichment followed by strand specific RNA-Seq library preparation (Ultra II Directional RNA Library Prep, NEB) following the manufacturer's instructions. Libraries were equimolarly pooled and subjected to 76 bp single end sequencing on a NextSeq 500 and Hiseq 2500 sequencer (Illumina) resulting in on average 29 Mio reads/sample (Bray et al., 2016; Kim et al., 2013) .
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