Nebnext library prep kit, by New England Biolabs - Product details

1

Transcriptomic Profiling of Peromyscus Tissues

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Liver and testis tissues were obtained (and frozen immediately in liquid nitrogen) from scrotal males of four species (P. attwateri, P. boylii, P. leucopus, and P. maniculatus) during the summer of 2012, 2013, or 2014 and archived at the Natural Science Research Laboratory, Museum of Texas Tech University (table 1). Tissues were separately homogenized and RNA extracted using the TRIzol reagent (ThermoFisher) following the manufacturer’s recommended protocol. RNA quality was quantified using a Bioanalyzer System (Agilent Genomics) with a minimum of 7.5 for the RNA Integrity Number score. Sequencing libraries were generated from 500 ng of RNA from each sample using the NEB NEXT library prep kit (New England Biolabs, Beverly, MA). Indexed libraries were pooled in equimolar concentrations and paired-end, 100-bp reads were sequenced from the cDNA libraries using an Illumina HiSeq 2000. Illumina paired-end reads were clipped, trimmed, and orphans were sorted using Trimmomatic v0.27 (Bolger et al. 2014 (link)). To count the number of pairs and improper or orphaned read alignments, reads were aligned using the program, Bowtie2 v2.3.4 (Langmead and Salzberg 2012 (link)). In total, eight transcriptomes were generated, two for each individual, representing the liver and testis samples.

Lindsey L.L., Platt RN I.I., Phillips C.D., Ray D.A, & Bradley R.D. (2020). Differential Expression in Testis and Liver Transcriptomes from Four Species of Peromyscus (Rodentia: Cricetidae). Genome Biology and Evolution, 12(1), 3698-3709.

+ Open protocol

+ Expand

2

Profiling Histone Modifications by ChIP-Seq

Cited 3 times

Check if the same lab product or an alternative is used in the 5 most similar protocols

ChIP assays were performed using protocols described (Arda et al., 2016 (link); Rada-Iglesias et al., 2011 (link); Wapinski et al., 2013 (link)). On average 2.5×10⁵ sorted cells were used for each ChIP-Seq experiment. 0.5 mg of the following antibodies were used to immunoprecipitate modified histone proteins: anti-H3K4me3 (Abcam, ab8580), anti-H3K4me1 (Abcam, ab8895), anti-H3K27me3 (Diagenode pAb-069–010), anti-H3K27ac (Abcam, ab4729). NEBNext library prep kit (NEB, E6240) was used to prepare sequencing libraries of ChIP fragments. Barcoded ChIP-Seq and input libraries were multiplexed and sequenced as single-end 50 bp reads on Illumina HiSeq2000 platform.

Efsun Arda H., Tsai J., Rosli Y.R., Giresi P., Bottino R., Greenleaf W.J., Chang H.Y, & Kim S.K. (2018). A chromatin basis for cell lineage and disease risk in the human pancreas. Cell systems, 7(3), 310-322.e4.

+ Open protocol

+ Expand

3

Transcriptional Profiling of N-myc Subsets in Hematopoietic Stem Cells

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cell populations were sorted directly into Trizol reagent (Life Technologies). Nucleic acids were extracted according to manufacturer instructions and precipitated in isopropanol with glycogen or linear acrylamide (Ambion) used as a carrier. After 30 min of DNase I treatment (Life Technologies), RNA was purified with RNA clean and concentrator columns (Zymo). RNA integrity was verified on a Agilent 2100 Bioanalyzer Pico chip. For small-scale RNA sequencing, full-length cDNA libraries were prepared using the SMARTer Ultra Low Input RNA kit (Clontech). Amplified cDNA libraries were sheared to 200–500 bp fragments using a Covaris LE220. End repair, A-tailing and Illumina adaptor ligation reactions were carried out using a NEBNext Library Prep Kit (New England BioLabs). Libraries were sequenced on an Illumina HiSeq2500 v4 using 50-bp paired-end reads. Fastq files were aligned to mm9 using TopHat allowing 2 mismatches. Aligned features were sorted with samtools, counted with htseq-count and differential expression was determined using the ‘edgeR’ package in Bioconductor, as described⁵⁵. Microarray gene expression profiling of sorted N-myc^hi and N-myc^lo HSC and GSEA was carried out as previously described^{27 (link)}. Heat map representations of row mean centered expression values were generated using the ‘pheatmap’ package in Bioconductor.

King B., Boccalatte F., Moran-Crusio K., Wolf E., Wang J., Kayembe C., Lazaris C., Yu X., Aranda-Orgilles B., Lasorella A, & Aifantis I. (2016). The ubiquitin ligase HUWE1 regulates hematopoietic stem cell maintenance and lymphoid commitment. Nature immunology, 17(11), 1312-1321.

+ Open protocol

+ Expand

4

RNA-seq Library Preparation and Analysis

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total RNA was prepared using the Qiagen RNeasy kit. Libraries were prepared using the NEBNext Library Prep Kit (New England Biolabs) according to the manufacturer's instructions. Library quality was assessed using a Bioanalyzer (Agilent) and then the samples were sequenced on the Illumina Hiseq 2000 with a goal of 30 million reads per sample. Raw FASTQ files were aligned using PRADA and FPKM values obtained using Cuffllinks for gene expression analysis.

Zhu K., Sun J., Kang Z., Zou Z., Wu G, & Wang J. (2017). Electroacupuncture Promotes Remyelination after Cuprizone Treatment by Enhancing Myelin Debris Clearance. Frontiers in Neuroscience, 10, 613.

+ Open protocol

+ Expand

5

Library Preparation for Sequencing

Check if the same lab product or an alternative is used in the 5 most similar protocols

After assessing enrichment at positive control sites using qPCR, we prepared libraries using the New England Biolabs NEBNext library prep kit (E6240L, New England Biolabs, Ipswich, MA, USA), with some changes. We performed adapter ligation before size selection, used Bioo adapters (514103, Bioo Scientific, Austin, TX, USA), and Ampure XP beads (A63881, Beckman-Coulter, Brea, CA, USA) instead of columns for all purifications and size selections. Sequencing was performed on a HiSeq 2500 in either the Genome Sequencing Facility at MD Anderson Cancer Center at Science Park (Smithville, TX) or the UT Austin Genome Sequencing and Analysis Facility (GSAF).

Hall A.W., Battenhouse A.M., Shivram H., Morris A.R., Cowperthwaite M.C., Shpak M, & Iyer V.R. (2018). Bivalent chromatin domains in glioblastoma reveal a subtype-specific signature of glioma stem cells. Cancer research, 78(10), 2463-2474.

+ Open protocol

+ Expand

6

Investigating TEPP-46 Effects on HK-2 Cells

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cultured HK-2 cells were obtained from Beijing Beina Chuanglian Biotechnology Research Institute, where they were cultured in Dulbecco’s modified eagle medium with low glucose (Sigma-Aldrich Inc., St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Sigma-Aldrich, St. Louis, MO, USA), 10 units/ml penicillin, and 10 mg/ml streptomycin and maintained in a continuous culture at 37°C in a humidified atmosphere (5% CO₂) in an incubator. The growth medium was shifted every 2 or 3 days, and the cells were sub-cultured until further measurements at 80% colony confluency. The obtained HK-2 cells were exposed to 25 mM HG medium as the hyperglycemic condition for 7 days. Thereafter, three replicates of cells exposed to HG were separately treated with (case group) or without (control group) 10 μM TEPP-46 for 1 day. We extracted 2 μg RNA per cell replicate sample for the RNA sample preparation, generated the sequencing libraries using NEBNext Library Prep Kit (NEB, USA) following the manufacturer’s recommendations, and sequenced these on the Illumina Hiseq X Ten platform to generate the 150-bp paired-end reads step by step.

Wang Z., Yu J., Hao D., Liu X, & Wang X. (2022). Transcriptomic signatures responding to PKM2 activator TEPP-46 in the hyperglycemic human renal proximal epithelial tubular cells. Frontiers in Endocrinology, 13, 965379.

+ Open protocol

+ Expand

7

RNA-seq Library Preparation and Sequencing

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total RNA was prepared using the Qiagen RNeasy kit. Libraries were prepared using the NEBNext library Prep Kit (New England Biolabs) according to the manufacturer’s instructions. Library quality was assessed using a Bioanalyzer (Agilent) and then were sequenced on the Illumina Hiseq 2000 with a goal of 30 million reads per sample. Raw FASTQ files were aligned using PRADA and FPKM values obtained using Cufflinks for gene expression analysis.

Wang J., Mikse O., Liao R.G., Li Y., Tan L., Janne P.A., Gray N.S., Wong K.K, & Hammerman P.S. (2014). Ligand associated ERBB2/3 activation confers acquired resistance to FGFR inhibition in FGFR3 dependent cancer cells. Oncogene, 34(17), 2167-2177.

+ Open protocol

+ Expand

8

RNA-seq Library Preparation and Sequencing

Check if the same lab product or an alternative is used in the 5 most similar protocols

Total RNA was prepared using the Qiagen RNeasy kit. Libraries were prepared using the NEBNext library Prep Kit (New England Biolabs) according to the manufacturer’s instructions. Library quality was assessed using a Bioanalyzer (Agilent) and then were sequenced on the Illumina Hiseq 2000 with a goal of 30 million reads per sample. Raw FASTQ files were aligned using PRADA and FPKM values obtained using Cufflinks for gene expression analysis.

Wang J., Mikse O., Liao R.G., Li Y., Tan L., Janne P.A., Gray N.S., Wong K.K, & Hammerman P.S. (2014). Ligand associated ERBB2/3 activation confers acquired resistance to FGFR inhibition in FGFR3 dependent cancer cells. Oncogene, 34(17), 2167-2177.

+ Open protocol

+ Expand

9

Transcriptional Profiling of N-myc Subsets in Hematopoietic Stem Cells

Cited 1 time

Check if the same lab product or an alternative is used in the 5 most similar protocols

Cell populations were sorted directly into Trizol reagent (Life Technologies). Nucleic acids were extracted according to manufacturer instructions and precipitated in isopropanol with glycogen or linear acrylamide (Ambion) used as a carrier. After 30 min of DNase I treatment (Life Technologies), RNA was purified with RNA clean and concentrator columns (Zymo). RNA integrity was verified on a Agilent 2100 Bioanalyzer Pico chip. For small-scale RNA sequencing, full-length cDNA libraries were prepared using the SMARTer Ultra Low Input RNA kit (Clontech). Amplified cDNA libraries were sheared to 200–500 bp fragments using a Covaris LE220. End repair, A-tailing and Illumina adaptor ligation reactions were carried out using a NEBNext Library Prep Kit (New England BioLabs). Libraries were sequenced on an Illumina HiSeq2500 v4 using 50-bp paired-end reads. Fastq files were aligned to mm9 using TopHat allowing 2 mismatches. Aligned features were sorted with samtools, counted with htseq-count and differential expression was determined using the ‘edgeR’ package in Bioconductor, as described⁵⁵. Microarray gene expression profiling of sorted N-myc^hi and N-myc^lo HSC and GSEA was carried out as previously described^{27 (link)}. Heat map representations of row mean centered expression values were generated using the ‘pheatmap’ package in Bioconductor.

King B., Boccalatte F., Moran-Crusio K., Wolf E., Wang J., Kayembe C., Lazaris C., Yu X., Aranda-Orgilles B., Lasorella A, & Aifantis I. (2016). The ubiquitin ligase HUWE1 regulates hematopoietic stem cell maintenance and lymphoid commitment. Nature immunology, 17(11), 1312-1321.

+ Open protocol

+ Expand

10

Single-Cell RNA Sequencing Protocol with Hydrogel Droplets

Check if the same lab product or an alternative is used in the 5 most similar protocols

Single-cell hydrogel droplets were retrieved from the microwells on the COC plate, then a chelating buffer (55mM sodium citrate, 30mM EDTA, 150mM NaCl) was introduced to depolymerize the hydrogels containing single-cells. RT reactions were performed as described previously to generate cDNA for library construction^{23 , 30 (link)}. For each single-cell sample, cDNA was obtained with REPLI-g WTA Single Cell Kit (Qiagen) according to the manufacturer’s protocol. Fragmentation of amplified cDNA was performed with NEBNext dsDNA Fragmentase (NEB). For each sample, 100 ng of small fragments (50−500bp) were used for RNA-Seq library preparation with the NEBNext Library Prep kit (NEB). All libraries were quantified using BioAnalyzer 2100 (Agilent) and KAPA Library Quantification Standards Kits for Illumina platforms (KAPA) and were sequenced on the Illumina HiSeq 2000 platform (Illumina, USA).

Zhang B., Xu H., Huang Y., Shu W., Feng H., Cai J., Zhong J.F, & Chen Y. (2019). Improving single-cell transcriptome sequencing efficiency with a microfluidic phase-switch device. The Analyst, 144(24), 7185-7191.

+ Open protocol

+ Expand

Nebnext library prep kit

Lab products found in correlation

Close spelling variants of this product

10 protocols using nebnext library prep kit

Transcriptomic Profiling of Peromyscus Tissues

Profiling Histone Modifications by ChIP-Seq

Transcriptional Profiling of N-myc Subsets in Hematopoietic Stem Cells

RNA-seq Library Preparation and Analysis

Library Preparation for Sequencing

Investigating TEPP-46 Effects on HK-2 Cells

RNA-seq Library Preparation and Sequencing

RNA-seq Library Preparation and Sequencing

Transcriptional Profiling of N-myc Subsets in Hematopoietic Stem Cells

Single-Cell RNA Sequencing Protocol with Hydrogel Droplets

About PubCompare

Ready to get started?