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Nebnext ultra 2 directional library prep kit for

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The NEBNext Ultra II Directional Library Prep Kit for Illumina is a laboratory equipment product designed for preparing DNA samples for sequencing on Illumina platforms. It enables the generation of high-quality sequencing libraries from a variety of input materials, including genomic DNA and cDNA.

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Lab products found in correlation

Nebnext poly a mrna magnetic isolation module, by New England Biolabs (5 mentions) Trizol, by Thermo Fisher Scientific (5 mentions) Hs rna screentape, by Agilent Technologies (4 mentions) Novaseq 6000 system, by Illumina (4 mentions) Nebnext lib quant kit, by New England Biolabs (3 mentions) Nextseq 550, by Illumina (3 mentions) Rneasy mini isolation kit, by Qiagen (2 mentions) Nebnext rrna depletion kit, by New England Biolabs (2 mentions) Novaseq 6000, by Illumina (2 mentions) Nextseq 500, by Illumina (2 mentions) Turbo dna free kit, by Thermo Fisher Scientific (2 mentions) Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions) Agilent rna 6000 nano kit, by Agilent Technologies (1 mentions) Rneasy mini kit, by Qiagen (1 mentions) 2100 bioanalyzer, by Agilent Technologies (1 mentions) Agencourt ampure xp bead, by Beckman Coulter (1 mentions) Rneasy kit, by Qiagen (1 mentions) Dynabeads oligo dt 25, by Thermo Fisher Scientific (1 mentions) Nebnext library quant kit for, by Illumina (1 mentions) Minelute pcr purification kit, by Qiagen (1 mentions)

14 protocols using nebnext ultra 2 directional library prep kit for

1

Strand-Specific RNA-Seq Library Prep

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The NEBNext Ultra II Directional Library Prep Kit for Illumina (#NEBE7760L; New England Biolabs Inc., Ipswich, MA) was used on 25 ng of total RNA for the preparation of strand-specific sequencing libraries from each TRAP-isolated RNA sample (input, negative fraction, and positive fraction) and from conventionally isolated RNA samples from brain (tissue), according to manufacturer’s instructions. Briefly, polyA containing mRNA was purified using oligo-dT attached magnetic beads. mRNA was chemically fragmented and cDNA synthesized. For strand-specificity, the incorporation of dUTP instead of dTTP in the second strand cDNA synthesis does not allow amplification past this dUTP with the polymerase. Following cDNA synthesis, each product underwent end repair process, the addition of a single ‘A’ base, and finally ligation of adapters. The cDNA products were further purified and enriched using PCR to make the final library for sequencing. Library sizing was performed with HSRNA ScreenTape (#5067–5579; Agilent Technologies) and libraries were quantified by qPCR (Kappa Biosystems, Inc., Wilmington, MA). The libraries for each sample were pooled at 4 nM concentration and sequenced using an Illumina NovaSeq 6000 system (SP PE50bp) at the Oklahoma Medical Research Foundation Genomics Facility.
Chucair-Elliott A.J., Ocañas S.R., Stanford D.R., Ansere V.A., Buettner K.B., Porter H., Eliason N.L., Reid J.J., Sharpe A.L., Stout M.B., Beckstead M.J., Miller B.F., Richardson A, & Freeman W.M. (2020). Inducible cell-specific mouse models for paired epigenetic and transcriptomic studies of microglia and astroglia. Communications Biology, 3, 693.
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2

RNA-seq analysis of Nutlin-3a response

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IMR90 cells at PD28 were treated with DMSO for 9 hours, or Nutlin-3a for 6, 9, or 12 hours. For RNA isolation, cells were lysed in TRIzol (15596018, Thermo Fisher Scientific) and snap frozen. RNA was then isolated with chloroform extraction, followed by QIAGEN RNeasy Mini Kit isolation (#74106), including DNA digestion with DNAse. Poly(A) + RNA was then isolated using double selection with poly-dT beads (E7490, NEB), and RNA-seq libraries were prepared using a NEB-Next Ultra II Directional Library Prep Kit for Illumina (E7760, NEB). Library sizes were determined on a Bioanalyzer, and concentration determined using NEBNext Lib Quant Kit (E7630, NEB). Libraries were sequenced on an Illumina NextSeq 550, using paired-end sequencing of 42 bases per read.
Alexander K.A., Coté A., Nguyen S.C., Zhang L., Gholamalamdari O., Agudelo-Garcia P., Lin-Shiao E., Tanim K.M., Lim J., Biddle N., Dunagin M.C., Good C.R., Mendoza M.R., Little S.C., Belmont A., Joyce E.F., Raj A, & Berger S.L. (2021). p53 mediates target gene association with nuclear speckles for amplified RNA expression. Molecular cell, 81(8), 1666-1681.e6.
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3

ATAC-seq and RNA-seq of Myonuclei

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ATAC-sequencing transposition reaction and library preparation was performed on 78,000 PCM1+ve sorted myonuclei using published protocols57 (link). Libraries were sequenced for 150 cycles, 120 million reads, paired-end sequencing on a Novaseq 6000 (Illumina) at the Victorian Clinical Genetics Service (VCGS, Melbourne, Australia). For RNA-sequencing from sorted nuclei, ribosomal RNA was depleted from total RNA using NEBNext rRNA Depletion kit (E6310, New England Biolabs) before generation of sequencing libraries using NEBNext Ultra II Directional Library Prep Kit for Illumina (E7760, New England Biolabs). Library fragment sizes were selected at 200–400 bp in length according to manufacturer’s protocol. Libraries were sequenced for 150 cycles, 40 million reads, paired-end-sequencing on a Novaseq 6000 (Illumina) at the VCGS, Melbourne, Australia.
Watt K.I., Henstridge D.C., Ziemann M., Sim C.B., Montgomery M.K., Samocha-Bonet D., Parker B.L., Dodd G.T., Bond S.T., Salmi T.M., Lee R.S., Thomson R.E., Hagg A., Davey J.R., Qian H., Koopman R., El-Osta A., Greenfield J.R., Watt M.J., Febbraio M.A., Drew B.G., Cox A.G., Porrello E.R., Harvey K.F, & Gregorevic P. (2021). Yap regulates skeletal muscle fatty acid oxidation and adiposity in metabolic disease. Nature Communications, 12, 2887.
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4

Transcriptome Library Preparation from Total RNA

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One million pelleted cells were used for total RNA isolation with RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol. TURBO DNA-free Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for DNA traces removal. RNA Integrity Number was determined with BioAnalyzer 2100 instrument (Agilent, Santa Clara, CA, USA) using an Agilent RNA Nano 6000 Kit (Agilent, USA). The average RNA Integrity Number was 8. The initial amount for transcriptome library preparation was 500 ng of total RNA. The NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, Ipswich, MA, USA) was used for poly(A) RNA enrichment. Transcriptome libraries were constructed with NEBNext Ultra II Directional Library Prep Kit for Illumina (New England Biolabs, USA) and NEBNext Multiplex Oligos (Dual Index Primers Set 1) for Illumina (New England Biolabs, USA). Paired-end libraries were sequenced on the Illumina NovaSeq 6000 instrument with 2 × 100 cycles (Illumina, San Diego, CA, USA).
Lebedeva O.S., Sharova E.I., Grekhnev D.A., Skorodumova L.O., Kopylova I.V., Vassina E.M., Oshkolova A., Novikova I.V., Krisanova A.V., Olekhnovich E.I., Vigont V.A., Kaznacheyeva E.V., Bogomazova A.N, & Lagarkova M.A. (2023). An Efficient 2D Protocol for Differentiation of iPSCs into Mature Postmitotic Dopaminergic Neurons: Application for Modeling Parkinson’s Disease. International Journal of Molecular Sciences, 24(8), 7297.
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5

RNA-Seq Library Preparation Protocol

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Strand-specific libraries for RNA sequencing were prepared using the NEBNext Ultra II Directional Library Prep Kit for Illumina (New England Biolabs Inc., Ipswich, MA) according to previously described methods (Chucair-Elliott et al., 2019 ) with n=5/group samples randomly selected from the whole cohort of mice. Using 25ng of isolated total RNA from each sample, mRNA was purified using Oligo dT-attached magnetic beads. Isolated mRNA was fragmented and primed for first and second strand cDNA synthesis successively. For strand specificity, the incorporation of dUTP instead of dTTP in the second strand cDNA synthesis does not allow amplification past this dUTP with the polymerase. Using Agencourt Ampure XP beads (Beckman Coulter Life Sciences, Indianapolis, IN), the prepared double-stranded cDNA was purified for end repair, the addition of a single ‘A’ base and adapter ligation. The adapter ligated cDNA products were further purified and enriched using PCR to make the final library for subsequent sequencing. The libraries were sized using HS DNA ScreenTape (Agilent Technologies) and quantified using Qubit dsDNA HS assay kit (Thermofisher Scientific). Libraries for each sample were normalized and pooled at 4nM and sequenced using an Illumina NovaSeq 6000 system (SP, PE50bp).
Ansere V.A., Ali-Mondal S., Sathiaseelan R., Garcia D.N., Isola J.V., Henseb J.D., Saccon T.D., Ocañas S.R., Tooley K.B., Stout M.B., Schneider A, & Freeman W.M. (2020). Cellular hallmarks of aging emerge in the ovary prior to primordial follicle depletion. Mechanisms of ageing and development, 194, 111425.
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6

RNA Extraction and Sequencing from PDX Samples

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According to the manufacturer’s guidelines, the RNA extraction was performed from PDX’s frozen fragment (25–30 mg) of cellular pellet using RNeasy Kit (74106, Qiagen). The RNAs were processed using the NEB Next Ultra II Directional Library Prep Kit for Illumina (E7765, NEB) and sequenced on the Illumina NextSeq500 with single-end, 75-base-pair-long reads.
Bolis M., Bossi D., Vallerga A., Ceserani V., Cavalli M., Impellizzieri D., Di Rito L., Zoni E., Mosole S., Elia A.R., Rinaldi A., Pereira Mestre R., D’Antonio E., Ferrari M., Stoffel F., Jermini F., Gillessen S., Bubendorf L., Schraml P., Calcinotto A., Corey E., Moch H., Spahn M., Thalmann G., Kruithof-de Julio M., Rubin M.A, & Theurillat J.P. (2021). Dynamic prostate cancer transcriptome analysis delineates the trajectory to disease progression. Nature Communications, 12, 7033.
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7

RNA-Sequencing Library Preparation

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We used ribo-depleted total RNA for most of the RNA-sequencing in this paper. Only for a few cases, we used polyA+ RNA for sequencing, and we labeled them in the legends (e.g., Figs. 6b, 7). 5–200 ng RNA was used for each library preparation using NEBNext Ultra II Directional Library Prep Kit for Illumina (NEB, E7760) following the manufacturer’s instructions. Ribosome RNA was depleted with NEBNext rRNA Depletion Kit (NEB, E6301). For Poly-A RNA-Seq, the total RNAs were selected by NEBNext® Poly(A) mRNA Magnetic Isolation Module (NEB, E7490) and the resulting polyA RNAs were used for library making in the same way as above described. Generated libraries in this study were mostly sequenced using NextSeq 550 Sequencing System with paired-end 40nt/40nt mode following the manufacturer’s instructions.
Xiong F., Wang R., Lee J.H., Li S., Chen S.F., Liao Z., Hasani L.A., Nguyen P.T., Zhu X., Krakowiak J., Lee D.F., Han L., Tsai K.L., Liu Y, & Li W. (2021). RNA m6A modification orchestrates a LINE-1–host interaction that facilitates retrotransposition and contributes to long gene vulnerability. Cell Research, 31(8), 861-885.
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8

Strand-specific RNA sequencing using NEBNext Ultra II

Cited 1 time
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The NEBNext Ultra II Directional Library Prep Kit for Illumina (#NEBE7760L; New England Biolabs Inc., Ipswich, MA) was used with 25 ng of total RNA for the preparation of strand-specific sequencing libraries from TRAP-isolated RNA samples (input and positive fraction) according to manufacturer’s instructions (Chucair-Elliott et al., 2020 (link)). Briefly, polyA containing mRNA was purified using oligo-dT attached magnetic beads. mRNA was chemically fragmented and cDNA synthesized. For strand-specificity, the incorporation of dUTP instead of dTTP in the second strand cDNA synthesis does not allow amplification past this dUTP with the polymerase. Following cDNA synthesis, each product underwent end repair process, the addition of a single ‘A’ base, and finally ligation of adapters. The cDNA products were further purified and enriched using PCR to make the final library for sequencing. Library sizing was performed with HS RNA ScreenTape (#5067-5579; Agilent Technologies). The libraries for each sample were pooled at 2–4 nM concentration and sequenced using an Illumina NovaSeq 6000 system (PE150bp) at the OMRF Clinical Genomics Facility.
Chucair-Elliott A.J., Ocañas S.R., Pham K., Van Der Veldt M., Cheyney A., Stanford D., Gurley J., Elliott M.H, & Freeman W.M. (2022). Translatomic response of retinal Müller glia to acute and chronic stress. Neurobiology of disease, 175, 105931.
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9

Strand-specific RNA sequencing using NEBNext Ultra II

Cited 1 time
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The NEBNext Ultra II Directional Library Prep Kit for Illumina (#NEBE7760L; New England Biolabs Inc., Ipswich, MA) was used with 25 ng of total RNA for the preparation of strand-specific sequencing libraries from TRAP-isolated RNA samples (input and positive fraction) according to manufacturer’s instructions (Chucair-Elliott et al., 2020 (link)). Briefly, polyA containing mRNA was purified using oligo-dT attached magnetic beads. mRNA was chemically fragmented and cDNA synthesized. For strand-specificity, the incorporation of dUTP instead of dTTP in the second strand cDNA synthesis does not allow amplification past this dUTP with the polymerase. Following cDNA synthesis, each product underwent end repair process, the addition of a single ‘A’ base, and finally ligation of adapters. The cDNA products were further purified and enriched using PCR to make the final library for sequencing. Library sizing was performed with HS RNA ScreenTape (#5067-5579; Agilent Technologies). The libraries for each sample were pooled at 2–4 nM concentration and sequenced using an Illumina NovaSeq 6000 system (PE150bp) at the OMRF Clinical Genomics Facility.
Chucair-Elliott A.J., Ocañas S.R., Pham K., Van Der Veldt M., Cheyney A., Stanford D., Gurley J., Elliott M.H, & Freeman W.M. (2022). Translatomic response of retinal Müller glia to acute and chronic stress. Neurobiology of disease, 175, 105931.
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10

Small RNA and mRNA Sequencing Protocol

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Small RNA libraries were prepared using the NEBnext small RNA library kit for Illumina (New England Biolabs, Cat. No E7330S), following the standard protocol with the following parameters: 1) 1:2 dilution of 3’SR Adaptor, SR RT Primer, and 5’SR Adaptor, 2) 15 indexing PCR cycles, 3) cleanup of final libraries with QIAgen MinElute PCR purification kit (QIAgen, Cat. No 28004), followed by a second cleanup with 2X SPRI. Libraries were analyzed on 6% non-denaturing polyacrylamide gels.
For mRNA-seq libraries, polyA RNA was enriched from 2 μg total RNA using Dynabeads Oligo(dT)25 (Invitrogen, Cat. No 61002). polyA-enriched fraction was used for library construction using the NEBnext Ultra II Directional Library Prep Kit for Illumina (New England Biolabs, Cat. No. E7760L), following the standard protocol but using half the reaction volumes recommended. Libraries were analyzed on 2% agarose gels.
Both small RNA-seq and mRNA-seq libraries were quantified using NEBNext Library Quant Kit for Illumina (New England Biolabs, Cat. No E7630L). Libraries were sequenced on an Illumina NextSeq500.
Scacchetti A., Shields E.J., Trigg N.A., Wilusz J.E., Conine C.C, & Bonasio R. (2023). A ligation-independent sequencing method reveals tRNA-derived RNAs with blocked 3’ termini. bioRxiv.
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