Mouse BM neutrophils were enriched using a mouse neutrophil isolation kit (Miltenyi Biotec). Total RNAs were isolated using the RNeasy Plus Mini Kit (Qiagen), and 300 ng of total RNA was used to generate the RNA-seq library using the NEBNext Ultra II RNA Library Prep with Sample Purification Beads (NEB) according to the manufacturer’s instructions. Briefly, mRNAs were isolated from total RNAs with the NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB), fragmented, and primed. First-strand cDNAs were synthesized, following by second-strand synthesis. The ends of purified double-strand cDNAs were repaired, ligated with an adaptor, and amplified with barcoded primers (NEBNext Multiplex Oligos for Illumina) using PCR. The purified final PCR product was sequenced using Illumina NovaSeq 6000. The reads were aligned to the mouse transcriptome (mm10) using a default setting of STAR (v.020201). Aligned reads were counted using HTseq (v.0.6.1p1). GSEA was performed using inflammation-related gene sets (positive regulation of inflammatory response (gene ontology [GO]: 0050729) and negative regulation of inflammatory response (GO:0050728)) obtained from the GO database. The Gene Expression Omnibus accession no. is GSE172394 .
Nebnext ultra 2 rna library prep with sample purification beads
Manufactured by New England Biolabs
The NEBNext Ultra II RNA Library Prep with Sample Purification Beads is a kit that enables the preparation of RNA libraries for next-generation sequencing. The kit includes reagents and protocols for RNA fragmentation, cDNA synthesis, adapter ligation, and bead-based sample purification.
Automatically generated - may contain errors
Lab products found in correlation
Qubit dsdna br assay kit, by Thermo Fisher Scientific (5 mentions)
2100 bioanalyzer, by Agilent Technologies (5 mentions)
Nebnext poly a mrna magnetic isolation module, by New England Biolabs (4 mentions)
Novaseq 6000 platform, by Illumina (4 mentions)
Nebnext poly a magnetic isolation module, by New England Biolabs (2 mentions)
Mouse neutrophil isolation kit, by Miltenyi Biotec (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
Qscript cdna supermix, by Avantor (1 mentions)
Bioanalyzer rna pico assay, by Agilent Technologies (1 mentions)
Superscript vilo master mix, by Thermo Fisher Scientific (1 mentions)
Allprep dna rna mini kit, by Qiagen (1 mentions)
Novaseq 6000 lane, by Illumina (1 mentions)
7 protocols using nebnext ultra 2 rna library prep with sample purification beads
1
Mouse Neutrophil RNA-seq Profiling
2
Illumina RNA-seq Library Preparation
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The Illumina RNA-seq library was prepared in a 96-well plate. We purified and enriched mRNA from 1 ug of total RNA using the NEBNext Poly(A) Magnetic Isolation Module (New England Biolabs, catalog no. E7490L). RNA fragmentation, first and second strand cDNA synthesis, and end-repair processing was performed using the NEBNext Ultra II RNA Library Prep with Sample Purification Beads (New England Biolabs, catalog no. E7775L). We ligated adapters in the cDNA library using adapters and unique dual indexes from the NEBNext Multiplex Oligos for Illumina (New England Biolabs, catalog no. E6440L). All procedures were performed following the manufacturer protocols. We used Qubit dsDNA BR Assay Kit (Invitrogen, catalog no. Q32853) to determine the concentration of the RNA library. The library was then pooled and qualified using the 2100 Bioanalyzer (Agilent) at Novogene, CA, USA. We sequenced the pooled library with the Illumina NovaSeq 6000 platform (150-bp paired-end reads).
3
Comprehensive RNA Extraction and Analysis Protocol
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Total ribonucleic acid (RNA) was isolated using the Qiagen Allprep RNA/DNA mini kit (80204, Qiagen) or Qiagen RNeasy RNA micro kit (74004, Qiagen, Hilden, Germany). Complementary deoxyribonucleic acid was synthesized using qScript cDNA SuperMix (total RNA >100 ng; 95048–100, VWR International, Radnor, Pennsylvania) or SuperScript VILO Master Mix (total RNA <100 ng; 11754050, Thermo Fisher Scientific, Waltham, Massachusetts). Messenger RNA (mRNA) levels were quantified using real-time quantitative polymerase chain reaction (qPCR) normalized to TATA-BOX Binding Protein (TBP), as previously described (21 (link)). Total RNA quality was examined using the Bioanalyzer RNA Pico assay (Agilent, Santa Clara, California). RNA-Seq libraries were prepared using the NEBNext Ultra II RNA Library Prep with Sample Purification Beads (7775, NEB, Ipswich, Massachusetts).
4
Illumina RNA-seq Library Preparation
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The Illumina RNA-seq library was prepared in a 96-well plate. We purified and enriched mRNA from 1 ug of total RNA using the NEBNext Poly(A) Magnetic Isolation Module (New England Biolabs, catalog no. E7490L). RNA fragmentation, first and second strand cDNA synthesis, and end-repair processing was performed using the NEBNext Ultra II RNA Library Prep with Sample Purification Beads (New England Biolabs, catalog no. E7775L). We ligated adapters in the cDNA library using adapters and unique dual indexes from the NEBNext Multiplex Oligos for Illumina (New England Biolabs, catalog no. E6440L). All procedures were performed following the manufacturer protocols. We used Qubit dsDNA BR Assay Kit (Invitrogen, catalog no. Q32853) to determine the concentration of the RNA library. The library was then pooled and qualified using the 2100 Bioanalyzer (Agilent) at Novogene, CA, USA. We sequenced the pooled library with the Illumina NovaSeq 6000 platform (150-bp paired-end reads).
5
Multiplexed RNA-seq Library Preparation
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RNAseq libraries for JU1373 and NIC58 were prepared simultaneously from mRNA isolated from 1 µg of pooled total RNA using the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs). RNA fragmentation, first and second strand cDNA synthesis, and end-repair processing were performed with the NEBNext Ultra II RNA Library Prep with Sample Purification Beads (New England Biolabs). Adapters and unique dual indexes in the NEBNext Multiplex Oligos for Illumina (New England Biolabs) were ligated, and the concentration of each library was determined using Qubit dsDNA BR Assay Kit (Invitrogen). Libraries were pooled and qualified by Bioanalyzer 2100 (Agilent; Novogene, CA, USA), and 150 bp paired-end reads were sequenced on a single Illumina NovaSeq 6000 lane.
6
C. briggsae RNA-seq Library Preparation
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We prepared Illumina RNA-seq libraries of C. briggsae strains QX1410 and VX34 in one 96-well plate simultaneously. For each sample, the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, catalog no. E7490L) was used to purify and enrich mRNA from 1 µg of total RNA. We performed RNA fragmentation, first and second strand cDNA synthesis, and end-repair processing using the NEBNext Ultra II RNA Library Prep with Sample Purification Beads (New England Biolabs, catalog no. E7775L). Adapters and unique dual indexes in the NEBNext Multiplex Oligos for Illumina (New England Biolabs, catalog no. E6440L) were used to adapter-ligate the cDNA libraries. We performed all procedures according to manufacturer protocols. We determined the concentration of each RNA-seq library using Qubit dsDNA BR Assay Kit (Invitrogen, catalog no. Q32853). RNA-seq libraries were pooled and qualified with the 2100 Bioanalyzer (Agilent) at Novogene, CA, USA. The pooled libraries were sequenced on a single lane of an Illumina NovaSeq 6000 platform, yielding 150-bp paired-end (PE150) reads.
7
Illumina RNA-seq Library Preparation
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We prepared Illumina RNA-seq libraries of C. briggsae strains QX1410 and VX34 in one 96-well plate simultaneously. For each sample, the NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, catalog no. E7490L) was used to purify and enrich mRNA from 1 µg of total RNA. We performed RNA fragmentation, first and second strand cDNA synthesis, and end-repair processing using the NEBNext Ultra II RNA Library Prep with Sample Purification Beads (New England Biolabs, catalog no. E7775L). Adapters and unique dual indexes in the NEBNext Multiplex Oligos for Illumina (New England Biolabs, catalog no. E6440L) were used to adapter-ligate the cDNA libraries. We performed all procedures according to manufacturer protocols. We determined the concentration of each RNA-seq library using Qubit dsDNA BR Assay Kit (Invitrogen, catalog no. Q32853). RNA-seq libraries were pooled and qualified with the 2100 Bioanalyzer (Agilent) at Novogene, CA, USA. The pooled libraries were sequenced on a single lane of an Illumina NovaSeq 6000 platform, yielding 150-bp paired-end (PE150) reads.
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