The method for vector insertion site determination has been detailed elsewhere.57 (link) Genomic DNA from sorted cell populations was isolated with DNeasy Blood & Tissue Kits (QIAGEN, Germantown, MD, USA). A control sample with 19 known and confirmed integration sites was spiked into each sample at 2% of the final amount. DNA samples were subjected to random shearing with a E210 sonicator (Covaris, Woburn, MA, USA) (200 cycles/burst, Intensity 3, 65 s). After shearing, the DNA ends were repaired with NEBNext End Repair Module, and then a poly(A) tail was added with the NEBNext dA-Tailing Module (New England Biolabs, Ipswich, MA, USA). Then, the NEBNext Adaptor for Illumina was added, followed by a round of linear amplification with a biotinylated primer. The amplified product was then enriched using streptavidin Dynal M-270 beads (Thermo Fisher Scientific, Waltham, MA, USA) and amplified further with a final nest PCR. Samples were pooled and run on an E-gel and DNA ranging from ∼250 to 800 bp was extracted with a DNA Gel Extraction Kit (QIAGEN, Germantown, MD, USA) and analyzed with Mi-Seq (Illumina, San Diego, CA, USA). 100–1,000 ng of genomic DNA isolated from sorted subpopulations of PB was used for VIS analysis using a quantitative PCR.
Nebnext da tailing module
Manufactured by New England Biolabs
Sourced in United States, United Kingdom
The NEBNext dA-Tailing Module is a laboratory tool designed to add adenine (A) nucleotides to the 3' ends of DNA fragments. This process, known as dA-tailing, is a common step in various molecular biology workflows, including DNA library preparation for next-generation sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Nebnext end repair module, by New England Biolabs (12 mentions)
Ampure xp bead, by Beckman Coulter (4 mentions)
Blunt ta ligase master mix, by New England Biolabs (3 mentions)
Agencourt ampure xp bead, by Beckman Coulter (3 mentions)
Dynabeads myone streptavidin c1 beads, by Thermo Fisher Scientific (2 mentions)
Abasi enzyme, by New England Biolabs (2 mentions)
Phusion dna polymerase, by New England Biolabs (2 mentions)
Hiseq 2000, by Illumina (2 mentions)
Qiaamp dna micro kit, by Qiagen (2 mentions)
S2 sonicator, by Covaris (2 mentions)
Minelute gel extraction kit, by Qiagen (2 mentions)
Monarch dna gel extraction kit, by New England Biolabs (2 mentions)
Pgem t easy vector, by Promega (2 mentions)
Monarch plasmid miniprep kit, by New England Biolabs (2 mentions)
Dna gel extraction kit, by Qiagen (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
E210 sonicator, by Covaris (1 mentions)
Le220, by Covaris (1 mentions)
Agilent bioanalyzer, by Agilent Technologies (1 mentions)
Hiseq system, by Illumina (1 mentions)
17 protocols using nebnext da tailing module
1
Vector Integration Site Determination
2
DNA Fragmentation and Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA (1 ug) was sonicated to a 400 to 600 bp size range using a Covaris LE220 acoustic shearing device (Covaris, Woburn, MA, USA). Fragments were end-repaired using the NEBNext EndRepair Module (New England Biolabs, Ipswich, MA, USA) and A-tailed with the NEBNext dA-Tailing Module. Illumina adapters were added using the NEBNext Quick Ligation Module. Ligation products were purified with AMPure XP beads (Beckman Coulter Genomics, Danvers, MA, USA). Libraries were quantified by qPCR using the KAPA Library Quantification Kit for Illumina Libraries (Kapa Biosystems, Wilmington, MA, USA) and library profiles were assessed using a DNA High Sensitivity LabChip kit on an AgilentBioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Libraries were sequenced on an Illumina MiSeq instrument (San Diego, CA, USA) using paired 150 base read chemistry.
3
RNA-Seq of Macrophage Transcriptome
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Modified from previously published protocol44 (link), the total RNA of macrophages was extracted and purified using TRIzol (Invitrogen, Carlsbad, CA, USA) and RNeasy Mini Kit (Qiagen, Venlo, Netherlands). Ribosomal RNA was depleted using the Human/mouse/Rat RiboZero Magnetic Kit (Epicentre, Madison, WI, USA), and verified using the Agilent RNA 6000 Nano Kit. DNases were removed using Turbo DNase (Invitrogen), and the sample was purified using RNAClean XP beads. The first strand of cDNA was synthesized using Superscript III (Invitrogen). Complimentary second strand cDNA was synthesized with RNase H and Klenow fragment of DNA polymerase I (Invitrogen). The cDNA was sonicated into 150 base pair fragments using a Covaris S220 Focused-ultrasonicator and deoxyadenosine monophosphate was incorporated to the cDNA fragment using NEBNext dA-Tailing Module (New England Biolabs). The cDNA library was sequenced using the Illumina HiSeq system.
4
AbaSeq for 5hmC Profiling in PGCs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total DNA was isolated from 10,000 sorted PGCs using the QIAamp DNA Micro Kit (Qiagen). AbaSeq libraries for 5hmC profiling were constructed as previously described15 (link). In brief, genomic DNA was glucosylated, then digested by AbaSI enzyme (NEB). Biotinylated P1 adapters were ligated onto the AbaSI digested DNA then fragmented using a Covaris S2 sonicator (Covaris), following the manufacturer’s instructions. Sheared P1-ligated DNA was then captured by mixing with Dynabeads MyOne Streptavidin C1 beads (Life Technologies) according to the manufacturer’s specifications. End repair and dA-tailing were carried out on the beads by using the NEBNext End Repair Module (NEB) and the NEBNext dA-tailing Module (NEB) at 20°C and 37 °C respectively for 30 min. P2 adapters were ligated to the random sheared ends of the dA-tailed DNA. Finally, the entire DNA was amplified using the Phusion DNA polymerase (NEB) with the addition of 300 nM forward primer (PCR_I) and 300 nM reverse primers (PCR_IIpe) for 16 cycles. The libraries were purified using AMPure XP beads (Beckman-Coulter) and sequenced on the Illumina HiSeq 2000 instrument.
5
16S rRNA Metagenome Sequencing for MinION
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To construct the 16S rRNA metagenome sequencing library for the MinION, the isolated metagenomic DNA was amplified using the S-D-bact-0008-c-S20 (5′-AGRGTTYGATYMTGGCTCAG-3′) and S-D-bact-1391-a-A-17 (5′-GACGGGCGGTGWGTRCA-3′) primers for broad range amplification of the 16S rRNA gene. The 16S rRNA amplification was monitored using CFX96 Real-Time PCR (Bio-Rad, Hercules, CA, USA) up to the saturation point (2 times) and the amplified DNA was purified using the MinElute Gel Extraction Kit (Qiagen, Venlo, Netherlands) (Fig. S1B ). The purified amplicons were end-repaired using the NEBNext® End Repair Module (NEB, Ipswich, MA, USA), and subsequently dA-tailed using the NEBNext® dA-Tailing Module (NEB), based on the SQK-LSK208 protocol (Oxford Nanopore Technologies, Oxford, UK). For MinION sequencing, the dA-tailed amplicon adapters were ligated using the provided adapter mix from Oxford Nanopore Technologies and the DNA was purified. The sequencing data were deposited on the European Nucleotide Archive with accession code: PRJEB22027.
6
ERCC RNA Spike-In Protocol for Transcriptome Profiling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The following materials were used: “ERCC RNA Spike-In Mix 1” stock (#4456740, Ambion, Thermo), RNA storage solution (Ambion, Thermo), Triton-X 100 (molecular biology grade, Sigma), dNTP mix (Advantage UltraPure PCR Deoxynucleotide Mix 10 mM each dNTP, Clontech), RNAse inhibitor (Clontech), Superscript III enzyme and Superscript III first-strand buffer (Invitrogen, Thermo), ultrapure DTT (Invitrogen, Thermo), Betaine (Sigma), MgCl2 (Sigma), oligo-dT30V primer (IDT), template switch primer (IDT), cDNA amplification primer (IDT) (the sequences are given in Supplementary Fig. S30 ), 2X KAPA2G Robust HotStart ReadyMix (kapabiosystems), AMPure XP beads (Beckman Coulter), NEBNext End Repair Module (New England Biolabs, UK), NEBNext dA-Tailing Module (New England Biolabs, UK).
7
Amplicon Sequencing on MinION Using ONT Kit
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Genomic DNA Sequencing Kit SQK-MAP-005 was used to prepare the amplicon library to be loaded into the MinION™. Approximately 250 ng of amplicon DNA (0.25 pmol) was processed for end repair using the NEBNext End Repair Module (New England Biolabs), followed by purification using Agencourt AMPure XP beads (Beckman Coulter). Subsequently, according to the manufacturer’s instructions, we used 200 ng of the purified amplicon DNA (~0.2 pmol) to perform dA-tailing using the NEBNext dA-tailing module (New England Biolabs) with a total volume of 30 μL, and incubated the sample at 37 °C for 15 min. Fifty μl of Blunt/TA ligase master mix (New England Biolabs), 10 μL of adapter mix, and 2 μL of HP adapter were added to the 30 μl dA-tailed amplicon DNA. The reaction was incubated at 16 °C for 15 min. The adapter-ligated amplicon was recovered using Dynabeads® His-Tag (Life Technologies) and washed with washing buffer provided with the Genomic DNA Sequencing Kit SQK-MAP-005 (Oxford Nanopore Technologies). Finally, the sample was eluted from the Dynabeads® by adding 25 μL of elution buffer and incubating for 10 min at room temperature before pelleting in a magnetic rack.
8
Nanopore Sequencing of Barcoded Amplicons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Before starting library preparation, we purified, quantified and estimated the purity of samples (Nanodrop). Two mixes were then prepared, each pulling six barcoded amplicons at equal weight ratio. One microgram of each pool was diluted to 80 μl in nuclease-free water and prepared for sequencing. According to the ONT Sequencing protocol (SQK-MAP005), DNA was end-repaired with the NEBNext End Repair Module (New England Biolabs Inc.) and subsequently dA-tailed using the NEBNext dA-Tailing Module (New England Biolabs Inc.), prior to ligation of nanopore-specific adapters with Blunt/TA Ligase Master Mix (New England Biolabs Inc.). All purifications were performed with Agencourt AMPure XP beads (Beckman Coulter Inc.). Dynabeads His-Tag Isolation & Pulldown (Life Technologies) was used to elute the library in the pre-sequencing Mix. After the Platform QC run, sequencing mix (75 μl of 2X Running Buffer, 66 μl of nuclease-free water, 3 μl of Fuel Mix and 6 μl of the Pre-sequencing Mix) was loaded and the MAP_48Hr_Sequencing_Run.py protocol was started (MinION flowcell: FLO-MAP003; MinKNOW software: v48.2.14). During running, two reloads of the sequencing mix were performed and the run was stopped after 24 h.
9
End Repair and dA-Tailing of ds cDNA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
End-repair was performed using the NEBNext End Repair Module (New England BioLabs E6050; New England BioLabs, Ipswich, MA, USA) with 6 µl of 10× end-repair buffer and 3 µl of end-repair enzyme mix added to each of the 51 µl ds cDNA samples, followed by incubation at room temperature for 25 min and clean-up using a 1.8× volume of Agencourt beads (as above), with elution in 25 µl nuclease-free water. Next, the end-repaired ds cDNA was dA-tailed with the NEBNext dA-Tailing Module (New England BioLabs E6053; New England BioLabs, Ipswich, MA, USA), using 3 µl of 10× NEBNext dA-Tailing Reaction Buffer and 2 µl of A-tailing enzyme (Klenow Fragment (3′ → 5′ exo-)) and incubation at 37 °C for 30 min, followed by clean-up with 1.8× Agencourt beads (as above) and elution in 15 µl of nuclease-free water.
10
Illumina Paired-End Library Prep
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Illumina-compatible, amplification-free, paired-end libraries were constructed with inserts spanning from 600 to > 1,000 base pair (bp). A total of 600 ng of DNA was sheared in a 60 µl volume on a Covaris S2 for 1 cycle of 40 s with a duty cycle of 5%, cycles per burst of 200 and an intensity of 3. Fragmented DNA was then end-repaired using the NEBNext End Repair Module (New England Biolabs), size-selected with a 0.58× Hi Prep bead clean-up (GC Biotech) and followed by A-tailing using the NEBNext dA-Tailing Module (New England Biolabs) and ligation of adaptors using the Blunt/TA Ligase Master Mix (New England Biolabs). Three 1× bead clean-ups were then undertaken to remove all traces of adaptor dimers. Library quality control was performed by running an Agilent Bioanalyzer High Sensitivity Chip and quantified using the KAPA Library Quantification Kit for Illumina Platforms (KAPA Biosystems). Based on the quantitative PCR quantification, libraries were loaded at 9 pM on an Illumina MiSeq System and sequenced with 300 bp paired reads.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!