The library for cDNA sequencing with an Illumina sequencer was constructed using a standard protocol from the NEBNext Ultra RNA Library Prep Kit for Illumina (New England BioLabs). Approximately 100 µg of total RNA was used for mRNA isolation by NEBNext Oligo d(T)25 beads (skipping the second bead wash step). The first and second strands of cDNA were synthesized using ProtoScript II Reverse Transcriptase and NEBNext Second Strand Synthesis Enzyme Mix. Synthesized double-stranded cDNA was end-repaired using NEBNext End Prep Enzyme Mix and ligated with a NEBNext Adaptor for Illumina. After the USER enzyme treatment, cDNA was amplified by PCR with the following conditions (20 μL cDNA, 2.5 μL Index Primer, 2.5 μL Universal PCR Primer, 25 μL NEBNext Q5 Hot Start HiFi PCR Master Mix 2× ; 98 °C for 30 s and 12 cycles each of 98 °C for 10 s, 65 °C for 75 s and 65 °C for 5 min). The library for direct DNA sequencing with the MinION sequencer was constructed using purified HMW gDNA. The library was completed following 1D library protocol SQK-LSK108 (Oxford Nanopore Technologies). The DNA-Seq library for MiSeq was prepared using a Hyper Plus Kit (KAPA Biosystems).
Nebnext adaptor for
Manufactured by Illumina
The NEBNext Adaptor for Illumina is a lab equipment product designed to be compatible with Illumina sequencing platforms. It serves as an adapter that enables the ligation of DNA fragments to Illumina sequencing primers, facilitating the library preparation process for Illumina sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Sqk lsk108, by Oxford Nanopore (1 mentions)
End prep enzyme mix, by Illumina (1 mentions)
Hyperplus kit, by Roche (1 mentions)
Nebnext ultra rna library prep kit for, by Illumina (1 mentions)
Nebnext ultra dna library prep kit for, by Illumina (1 mentions)
Nebnext ultra dna library prep kit, by Illumina (1 mentions)
Hiseq 2500, by Illumina (1 mentions)
Le220 focused ultrasonicator, by Covaris (1 mentions)
User enzyme, by Illumina (1 mentions)
Nebnext ultra 2 fs dna library prep kit for, by Illumina (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
6 protocols using nebnext adaptor for
1
cDNA and DNA Library Preparation Protocols
2
DNA Library Preparation for Illumina Sequencing
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Libraries were prepared using
300 ng of fragmented DNA (∼200
bp) and the NEBNext ultra DNA library prep kit for Illumina (NEB),
according to the manufacturer’s protocol. In detail, 300 ng
of fragmented DNA was mixed with 3.0 μL of EndPrep enzyme mix
and 6.5 μL of 10× end repair reaction buffer. The mixture
was incubated in a thermocycler for 30 min at 20 °C and subsequently
for 30 min at 65 °C.
Adaptors were ligated by adding 15
μL of blunt/TA ligase master mix (NEB), 2.5 μL of NEBNext
adaptor for Illumina (NEB), and 1 μL of ligation enhancer (NEB)
to the end prep reaction mixture. These samples were handled according
to the manufacturer’s instructions for adaptor ligation in
the NEBNext ultra DNA library prep kit for Illumina (NEB). Thereafter,
adaptor-ligated DNA was size-selected using size selection of adaptor-ligated
DNA in the NEBNext ultra DNA library prep kit for Illumina (NEB).
In short, first, AMPure XP beads were used to remove the unwanted
large DNA fragments. Second, AMPure XP beads were used to bind the
DNA fragments of interest. The DNA fragments from the beads were diluted
into 28 μL of 10 mM Tris-HCl.
300 ng of fragmented DNA (∼200
bp) and the NEBNext ultra DNA library prep kit for Illumina (NEB),
according to the manufacturer’s protocol. In detail, 300 ng
of fragmented DNA was mixed with 3.0 μL of EndPrep enzyme mix
and 6.5 μL of 10× end repair reaction buffer. The mixture
was incubated in a thermocycler for 30 min at 20 °C and subsequently
for 30 min at 65 °C.
Adaptors were ligated by adding 15
μL of blunt/TA ligase master mix (NEB), 2.5 μL of NEBNext
adaptor for Illumina (NEB), and 1 μL of ligation enhancer (NEB)
to the end prep reaction mixture. These samples were handled according
to the manufacturer’s instructions for adaptor ligation in
the NEBNext ultra DNA library prep kit for Illumina (NEB). Thereafter,
adaptor-ligated DNA was size-selected using size selection of adaptor-ligated
DNA in the NEBNext ultra DNA library prep kit for Illumina (NEB).
In short, first, AMPure XP beads were used to remove the unwanted
large DNA fragments. Second, AMPure XP beads were used to bind the
DNA fragments of interest. The DNA fragments from the beads were diluted
into 28 μL of 10 mM Tris-HCl.
3
Targeted DNA Sequencing Workflow
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Two hundred and fifty ng genomic DNA from each sample was fragmented to 200‐bp lengths using the LE220 Focused ultrasonicator (Covaris). The End Prep Enzyme Mix (New England Biolabs) was used for fragment‐end repair and the NEBNext Adaptor for Illumina (New England Biolabs) was used for adaptor ligation. Adaptor‐ligated DNA fragments were amplified for nine cycles. Seven hundred and fifty ng of each PCR product was subjected to target capture with custom‐made probes, as described previously.12 The captured library was amplified over 11 cycles, and massive parallel sequencing of the isolated fragments was performed with the HiSeq2500 (Illumina,) using the paired‐end option.
4
DNA Fragments Adaptor Ligation Protocol
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The maximal amount of input material during PRIA is the adaptor ligation step. Based on the manufacturer's instruction, 100 ng of the DNA fragments were repaired by reacting them with the End Prep Enzyme Mix and End Repair Reaction Buffer at 20°C for 30 min and then at 65°C for 30 min. Subsequently, they were reacted with the Blunt/TA Ligase Master Mix, the NEBNext Adaptor for Illumina and Ligation Enhancer at 20°C for 15 min before finally being treated with the USER™ enzyme at 37°C for 15 min. The adaptor-containing fragments were labeled with PHPA to obtain chemically labeled fragments, after which they were denatured at 95°C for 10 min and immediately transferred to ice to acquire single stranded DNA.
5
Genomic DNA Preparation for GUIDE-Seq
6
Whole-Genome DNA Library Preparation
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Whole-genome DNA library preparation was prepared as described for TnSeq libraries with the following modifications. For each sample, total DNA was extracted from 500 μl of overnight culture, purified and fragmented as specified for TnSeq procedures. For the adapter ligation step, NEBnext Adaptor for Illumina (New England Biolabs) was used. All the subsequent steps were done as described for TnSeq library preparation above. Different variants of rescued pVCR94Sp were sequenced to characterise scars. The assembly workflow started by trimming the reads based on their quality and the presence of the TruSeq Illumina adapters using Trimmomatic version 0.36 with the parameters SLIDINGWINDOW:4:20 MINLEN:36 ILLUMINACLIP::2:30:15 (28 (link)). The quality of the reads was assessed with FastQC version 0.11.5 before and after trimming. De novo assemblies were then generated using SPAdes version 3.11.1 with the parameters -k 21 33 55 77 –careful –only-assembler (34 (link)). QUAST version 5.0.2 was used to evaluate the quality of the assemblies (35 (link)). Full description of sequenced strains and assembly statistics are available in Supplemental Table S3 .
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