Libraries were prepared using the NEBNext Ultra DNA Library Prep for Illumina kit (no. E7370) according to the manufacturer’s protocol. Briefly, input and ChIP-enriched DNA were subjected to end repair and the addition of ‘A’ bases to 3′ ends, ligation of the NEB adapter and USER excision. All purification steps were performed using AgenCourt AMPure XP beads (no. A63882, Beckman Coulter). Library amplification was performed by PCR using NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs, nos. E6440, E6442, E6444, E6446). Final libraries were analyzed using either an Agilent Bioanalyzer or Fragment analyzer High Sensitivity assay (no. 5067‐4626 or DNF‐474) to estimate the quantity and check size distribution, and were then quantified by quantitative PCR using the KAPA Library Quantification Kit (no. KK4835, KapaBiosystems). Libraries were sequenced 1 × 50 + 8 + 8 base pairs (bp) on Illumina’s NextSeq2000. Between 35 and 40 million reads were obtained per sample.
Nebnext multiplex oligos for 96 unique dual index primer pairs
Manufactured by Illumina
Sourced in United States
The NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) is a set of oligonucleotides designed for use with Illumina sequencing platforms. The product contains 96 unique dual index primer pairs that can be used to label DNA samples for multiplex sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Agencourt ampure xp bead, by Beckman Coulter (7 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (7 mentions)
Nebnext poly a mrna magnetic isolation module, by New England Biolabs (7 mentions)
Bioanalyzer high sensitivity dna assay, by Agilent Technologies (6 mentions)
Agilent bioanalyzer, by Agilent Technologies (2 mentions)
Agilent 4200 tapestation system, by Agilent Technologies (2 mentions)
Nebnext ultra 2 directional rna library prep kit, by Illumina (2 mentions)
Kapa library quantification kit, by Roche (1 mentions)
Nextseq 2000, by Illumina (1 mentions)
Nebnext ultra dna library prep for kit, by Illumina (1 mentions)
Tapestation 2200, by Agilent Technologies (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nebnext rrna depletion kit human mouse rat, by Illumina (1 mentions)
Nebnext ultra 2 directional rna library preparation kit, by Illumina (1 mentions)
Rna screentape, by Agilent Technologies (1 mentions)
Hiseq platform, by Illumina (1 mentions)
Nebnext poly a mrna magnetic isolation module, by Illumina (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Nextseq 550, by Illumina (1 mentions)
D5000 screentape, by Agilent Technologies (1 mentions)
18 protocols using nebnext multiplex oligos for 96 unique dual index primer pairs
1
DNA Library Preparation for Illumina Sequencing
2
ARTIC SARS-CoV-2 Library Prep Protocol
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Libraries were prepared using the NEBNext® ARTIC SARS-CoV-2 Library Prep Kit, following standard protocol with cDNA Amplicon and Ligation Bead Clean-ups (Version 3.0 7/21). Manual library normalisation was performed to ensure even sample coverage, based on the library’s DNA concentration and average size, as measured by the Qubit (Thermo Fisher Scientific, UK) and 2200 TapeStation (Agilent Technologies, USA). Paired-end sequencing was performed using the MiSeq reagent kit v2, with 2×250bp, and one water control on each run. NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs) were used.
3
RNA Sequencing of Hematopoietic Stem Cells
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RNA was isolated from HSCs using the RNeasy Mini Kit (Qiagen) following the manufacturer’s instructions. Sequencing of RNA samples was performed using Illumina’s next-generation sequencing methodology55 (link). In brief, total RNA was quantified and quality checked using an Agilent 4200 Tapestation System in combination with RNA ScreenTape (both Agilent Technologies). Libraries were prepared from 300 ng of input material (total RNA) using NEBNext Ultra II Directional RNA Library Preparation Kit in combination with NEBNext rRNA Depletion Kit (Human/Mouse/Rat) and NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs) following the manufacturer's instructions (New England Biolabs). Quantification and quality check of libraries was done using an Agilent 4200 Tapestation System and D1000 ScreenTapes. Libraries were pooled and sequenced in one NovaSeq6000 SP 100 cycle run (101 cycle/single-end/standard loading workflow mode). Sequence information was converted to FASTQ format using bcl2fastq v2.20.0.422. Sequencing resulted in 62 × 106 reads per sample on average.
4
Directional RNA-seq library preparation
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150 ng of total RNA per sample were utilized as input for mRNA enrichment procedure with ‘NEBNext® Poly(A) mRNA Magnetic Isolation Module’ (E7490L; New England Biolabs) followed by stranded cDNA library generation using “NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina” (E7760L; New England Biolabs). All steps were performed as recommended in the user manual E7760 (Version 1.0_02-2017; NEB) except that all reactions were downscaled to 2/3 of initial volumes.
cDNA libraries were barcoded by dual indexing approach, using “NEBNext Multiplex Oligos for Illumina–96 Unique Dual Index Primer Pairs” (6440S; New England Biolabs). All generated cDNA libraries were amplified with 10 cycles of final PCR.
One additional purification step was introduced at the end of the standard procedure, using 1.2x “Agencourt® AMPure® XP Beads” (#A63881; Beckman Coulter, Inc.). Fragment length distribution of individual libraries was monitored using “Bioanalyzer High Sensitivity DNA Assay” (5,067-4626; Agilent Technologies). Quantification of libraries was performed by use of the “Qubit® dsDNA HS Assay Kit” (Q32854; ThermoFisher Scientific).
cDNA libraries were barcoded by dual indexing approach, using “NEBNext Multiplex Oligos for Illumina–96 Unique Dual Index Primer Pairs” (6440S; New England Biolabs). All generated cDNA libraries were amplified with 10 cycles of final PCR.
One additional purification step was introduced at the end of the standard procedure, using 1.2x “Agencourt® AMPure® XP Beads” (#A63881; Beckman Coulter, Inc.). Fragment length distribution of individual libraries was monitored using “Bioanalyzer High Sensitivity DNA Assay” (5,067-4626; Agilent Technologies). Quantification of libraries was performed by use of the “Qubit® dsDNA HS Assay Kit” (Q32854; ThermoFisher Scientific).
5
RNA-sequencing from Dissected Tissues
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Dissected tissues were kept in TRI reagent and stored at −80° C prior to RNA-extraction. Total RNA extraction was performed as described previously (Hart et al. 2018 ). Total RNA was quantified by a Qubit Fluorometer, and quality was checked by an Agilent Bioanalyzer. Libraries were constructed with New England Biolabs NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490S), NEBNext Ultra II Directional RNA Library Prep Kit (E7765S), and NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs, E6440S) following the manufacturer's instructions. Library quality was analyzed on an Agilent Bioanalyzer (supplementary table S1, Supplementary Material online). Libraries were pooled and sequenced on an Illumina HiSeq platform (2 × 150 bp reads). We obtained a total of 1,439,700,457 reads across 20 samples (10 tissues × 2 replicates) (supplementary table S1, Supplementary Material online).
6
Viral Genomic Sequencing Protocol
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This protocol was applied to virus isolates propagated in vitro. Extracted nucleic acid was incubated with DNase I (Thermo Fisher Scientific AM2222) for 5 min at 37°C. After DNase treatment, the samples were purified using Agencourt RNA clean AMPure XP beads (Beckman Coulter A63987), following the manufacturer's guidelines, and quantified using the Qubit dsDNA HS Kit (Thermo Fisher Scientific Q32854). cDNA was synthesized using SuperScript III (Thermo Fisher Scientific 18080044) and a NEBNext Ultra II non-directional RNA second strand synthesis module (New England Biolabs E6111L), as per the manufacturer's guidelines.
Samples were further processed using the Kapa LTP library preparation kit for Illumina Platforms (Kapa Biosystems KK8232). Briefly, the cDNA was end-repaired and the protocol followed through to adapter ligation. At this stage, the samples were uniquely indexed using the NEBNext multiplex oligos for Illumina 96 unique dual index primer pairs (New England Biolabs E6442S), with 15 cycles of PCR performed.
All amplified libraries were quantified by a Qubit dsDNA HS Kit and run on the Agilent 4200 Tapestation system (Agilent G2991AA) using the high-sensitivity D5000 Screentape (Agilent 5067-5592) and high-sensitivity D5000 reagents (Agilent 5067-5593). Libraries were sequenced on an Illumina NextSeq 550 (Illumina SY-415-1002).
Samples were further processed using the Kapa LTP library preparation kit for Illumina Platforms (Kapa Biosystems KK8232). Briefly, the cDNA was end-repaired and the protocol followed through to adapter ligation. At this stage, the samples were uniquely indexed using the NEBNext multiplex oligos for Illumina 96 unique dual index primer pairs (New England Biolabs E6442S), with 15 cycles of PCR performed.
All amplified libraries were quantified by a Qubit dsDNA HS Kit and run on the Agilent 4200 Tapestation system (Agilent G2991AA) using the high-sensitivity D5000 Screentape (Agilent 5067-5592) and high-sensitivity D5000 reagents (Agilent 5067-5593). Libraries were sequenced on an Illumina NextSeq 550 (Illumina SY-415-1002).
7
Metagenomic Library Preparation for Illumina Sequencing
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Metagenomic libraries were prepared with a standard DNA input of 50 ng across all samples, using NEBNext® Ultra™ II FS DNA Library Prep Kit for Illumina (New England Biolabs, Cat. No. E7805), according to the manufacturer’s instructions. The reaction volumes were, however, scaled to a quarter of the recommended volumes for cost effectiveness. Barcoding and enrichment of libraries was carried out using NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs; New England Biolabs, Cat. No. E6440). Paired-end sequencing (2 × 151 bp reads) was carried out on the Illumina HiSeq4K platform with a minimum and average depth per sample of 2.4 Gb and 9.4Gb respectively.
8
BRD4-bound Chromatin Profiling in CXCR4 Knockout
9
Bacterial Ribosomal RNA Depletion and RNA-seq
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The concentration of total RNA was measured with an Invitrogen™ Qubit™ 3 Fluorometer and a Qubit™ RNA High Sensitivity (HS) Kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Quality was assessed using the Bioanalyzer 2100 instrument with an RNA Nano 6000 Kit (Agilent, Santa Clara, CA, USA). Next, total RNA was depleted of ribosomal RNA with a NEBNext® rRNA Depletion Kit v2 (Bacteria) (New England Biolabs, Ipswich, MA, USA) and Agencourt RNAClean XP beads (Beckman Coulter, Pasadena, CA, USA). Fragmentation and library preparation was then performed according to the manufacturer’s instructions using an NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina. To facilitate multiplex sequencing, NEBNext® Multiplex Oligos for Illumina® (96 Unique Dual Index Primer Pairs) (New England Biolabs, Ipswich, MA, USA) was used to barcode the reads. Libraries were further purified with Agencourt AMPure XP Reagent (Beckman Coulter, Pasadena, CA, USA), and the quality and quantity of the amplified cDNA were assessed with a DNA High Sensitivity Kit (Agilent, Santa Clara, CA, USA). Following this, they were pooled, denatured, and diluted to 1.8 nM. Single-end reads were sequenced with NextSeq 500/550 Mid Output Kit v2.5 (150 cycles) reagents (Illumina, San Diego, CA, USA).
10
RNA-seq Library Preparation Protocol
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Of the total RNA, 500 ng per sample was utilized as the input for the mRNA enrichment procedure with “NEBNext® Poly(A) mRNA Magnetic Isolation Module” (New England Biolabs, E7490L, Ipswich, MA, USA), followed by stranded cDNA library generation using “NEBNext® Ultra II Directional RNA Library Prep Kit for Illumina” (New England Biolabs, E7760L, Ipswich, MA, USA). All steps were performed as per recommended by the manufacturer, except that all reactions were downscaled to 2/3 of the initial volumes. Furthermore, one additional purification step was introduced at the end of the standard procedure, using 1× “Agencourt® AMPure® XP Beads” (Beckman Coulter, Inc., A63881 Brea, CA, USA). cDNA libraries were barcoded via a dual indexing approach, using “NEBNext Multiplex Oligos for Illumina, 96 Unique Dual Index Primer Pairs” (New England Biolabs, 6440S, Ipswich, MA, USA). All generated cDNA libraries were amplified with seven cycles of the final PCR. The fragment length distribution of individual libraries was monitored using “Bioanalyzer High Sensitivity DNA Assay” (Agilent Technologies, 5067-4626, Santa Clara, CA, USA). Quantification of the libraries was performed through use of the “Qubit® dsDNA HS Assay Kit” (Thermo Fisher Scientific, Q32854, Waltham, MA, USA).
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