As previously mentioned, for fosmid DNA isolation, clones were grown in 100-ml flasks containing LB-Cm broth medium supplemented with 2 μl/ml of CopyControl Fosmid Autoinduction solution (Epicentre) for higher DNA yields, followed by incubation at 37°C for 12 to 16 h and with continuous stirring at 150 rpm. Fosmid DNA was extracted using the FosmidMax DNA purification kit (Epicentre) according to the manufacturer’s instructions and was then subjected to restriction analysis using ApaI enzyme (Thermo Scientific) to determine if different DNA insert sequences were present. Subsequently, fosmids were sequenced on the Illumina NextSeq 500, with a NextSeq 500/550 High Output reagent kit v2 (300 cycles), in accordance with the standard Illumina sequencing protocols. The KneadData pipeline (v. 0.6.1) (http://huttenhower.sph.harvard.edu/kneaddata ) was used to perform quality trimming using Trimmomatic (v. 0.38) (42 (link)), and sequences mapping to the cloning host and vector sequences were removed using bmtagger (v. 3.101) (ftp://ftp.ncbi.nlm.nih.gov/pub/agarwala/bmtagger/ ). The remaining reads were assembled using Unicycler (v. 0.4.7) (43 (link)), annotated with Prokka (v. 1.13) (44 (link)), and antibiotic resistance was profiled using the Resistance Gene Identifier web portal (45 (link)).
Nextseq 500 550 high output reagent kit v2
Manufactured by Illumina
The NextSeq 500/550 High Output Reagent Kit v2 is a laboratory equipment product designed for use with the NextSeq 500 and NextSeq 550 sequencing systems. It provides the necessary reagents and consumables required for high-throughput DNA sequencing applications.
Automatically generated - may contain errors
Lab products found in correlation
Nextseq 500, by Illumina (10 mentions)
Nextera xt library preparation kit, by Illumina (4 mentions)
Nextseq 500 platform, by Illumina (3 mentions)
Nextera xt dna library preparation guide, by Illumina (3 mentions)
Nextera xt kit, by Illumina (3 mentions)
Chromium single cell 3 library, by 10x Genomics (2 mentions)
Deep well reaction module, by Bio-Rad (2 mentions)
Spriselect beads, by Beckman Coulter (2 mentions)
Dynabeads myone silane beads, by Thermo Fisher Scientific (2 mentions)
S2 instrument, by Covaris (2 mentions)
Spriselect beads, by Covaris (2 mentions)
C1000 touch thermal cycler, by Bio-Rad (2 mentions)
Miseq sequencing platform, by Illumina (2 mentions)
Fosmidmax dna purification kit, by Illumina (1 mentions)
Copycontrol fosmid autoinduction solution, by Illumina (1 mentions)
Apai enzyme, by Thermo Fisher Scientific (1 mentions)
Qiamp dna stool mini kit, by Qiagen (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Single cell 3 chip, by 10x Genomics (1 mentions)
Nextera xt dna library preparation kit, by Illumina (1 mentions)
14 protocols using nextseq 500 550 high output reagent kit v2
1
Fosmid DNA Isolation and Sequencing
2
Illumina NextSeq Sequencing and Data Preprocessing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
NextSeq libraries were sequenced on the Illumina NextSeq 500, with a NextSeq 500/550 High Output Reagent Kit v2 (300 cycles), in accordance with the standard Illumina sequencing protocols resulting in over 600 million read pairs. Raw reads were converted from FastQ to BAM format using Picard Tools (v. 2.7.1) and SAMtools (v. 1.5)38 (link) and duplicate reads were removed using Picard Tools (https://github.com/broadinstitute/picard ). Low-quality reads were removed using the trimBWAstyle.usingBam.pl script from the Bioinformatics Core at UC Davis Genome Center (https://github.com/genome/genome/blob/master/lib/perl/Genome/Site/TGI/Hmp/HmpSraProcess/trimBWAstyle.usingBam.pl ). Specifically, bases with a quality score less than Q30 were trimmed and resulting reads shorter than 105bp were discarded.
Panphlan (v. 1.2.2.2)39 (link) was used to build a pangenome database of all freely available fish genome sequences. Host contamination was identified by aligning the quality-trimmed whole-metagenome sequencing reads to this pangenome database with Bowtie2 (v. 2.2.9).40 (link) The resulting alignments were converted to BAM format and host reads removed by SAMtools before conversion to FastQ format using BEDtools.41 (link) FastQ files were converted to FastA using the fq2fa script packaged with IDBA-UD (v. 1.1.1).42 (link)
Panphlan (v. 1.2.2.2)39 (link) was used to build a pangenome database of all freely available fish genome sequences. Host contamination was identified by aligning the quality-trimmed whole-metagenome sequencing reads to this pangenome database with Bowtie2 (v. 2.2.9).40 (link) The resulting alignments were converted to BAM format and host reads removed by SAMtools before conversion to FastQ format using BEDtools.41 (link) FastQ files were converted to FastA using the fq2fa script packaged with IDBA-UD (v. 1.1.1).42 (link)
3
Microbial DNA Extraction and Shotgun Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was extracted using the QIAmp DNA stool minikit (Qiagen, Crawley, West Sussex, United Kingdom)40 (link) following the manufacturer’s instructions with minor modifications and stored at – 80 °C until its use. Prior to shotgun sequencing, a Qubit High Sensitivity DNA assay (BioSciences, Dublin, Ireland) was used to determine the total DNA concentration, and purity was assessed by the 260/280 and 260/230 absorbance ratios using a spectrophotometer NanoDrop ND-1000 (Thermo Fisher Scientific, Wilmington, Delaware). Paired-end sequencing libraries were prepared from the extracted DNA using the Illumina Nextera XTLibrary Preparation Kit (Illumina Inc., San Diego, California) followed by sequencing on the Illumina NextSeq 500, with a NextSeq 500/550 High Output Reagent kit v2 (300 cycles), in accordance with the standard Illumina sequencing protocols.
4
Single-Cell Transcriptome Profiling Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-cell suspensions were loaded onto 10x Genomics Single Cell 3′ Chips along with the reverse transcription (RT) mastermix per the manufacturer's protocol for the Chromium Single Cell 3′ Library (10x Genomics; PN-120233) to generate single-cell gel beads in emulsion (GEMs). Reverse transcription was performed using a C1000 Touch Thermal Cycler with a Deep Well Reaction Module (Bio-Rad) as follows: for 2 h at 55°C; for 5 min at 85°C; hold 4°C. cDNA was recovered and purified with DynaBeads MyOne Silane Beads (Thermo Fisher Scientific; catalog no. 37002D) and SPRIselect beads (Beckman Coulter; catalog no. B23318). Purified cDNA was amplified as follows: for 3 min at 98°C; 12× (for 15 sec at 98°C; for 20 sec at 67°C; for 60 sec at 72°C); for 60 sec at 72°C; hold 4°C. Amplified cDNA was purified using SPRIselect beads and sheared to ∼200 bp with a Covaris S2 instrument (Covaris) using the manufacturer's recommended parameters. Sequencing libraries were generated with unique sample indices (SI) for each sample. Libraries for samples 1–3 and 4–5 were multiplexed, respectively, and sequenced on an Illumina NextSeq 500 (NextSeq control software v2.0.2/Real Time Analysis v2.4.11) using a 150-cycle NextSeq 500/550 High Output Reagent Kit v2 (Illumina, FC-404-2002) in standalone mode as follows: 98 bp (Read 1), 14 bp (I7 Index), 8 bp (I5 Index), and 10 bp (Read 2).
5
Environmental DNA sequencing library preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Extracted environmental DNA was employed to prepare the 150 bp paired-end sequencing libraries using the Illumina Nextera XTLibrary Preparation Kit (Illumina Inc., San Diego, CA, USA) using the method described by Rinke et al. [44 ]. Sequencing was performed on the Illumina NextSeq 500 platform using a NextSeq 500/550 High Output Reagent kit v2 (300 cycles), in accordance with the standard Illumina sequencing protocols.
6
Whole-Metagenome Shotgun Library Prep
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Nextera XT DNA Library Preparation kit (Illumina) was used for the preparation of whole-metagenome shotgun libraries. NextSeq libraries were sequenced on the Illumina NextSeq 500, with a NextSeq 500/550 High Output Reagent Kit v2 (300 cycles), in accordance with the standard Illumina sequencing protocols resulting in over 600 million read pairs.
7
Single-Cell RNA Sequencing Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Single-cell suspensions were loaded onto 10X Genomics Single Cell 3′ Chips along with the RT mastermix as per the manufacturer’s protocol for the Chromium Single Cell 3′ Library (v2, PN-120233; 10X Genomics), to generate single-cell gel beads in emulsion. RT was performed using a C1000 Touch Thermal Cycler with a Deep Well Reaction Module (Bio-Rad) as follows: 55°C for 2 h; 85°C for 5 min; hold 4°C. cDNA was recovered and purified with DynaBeads MyOne Silane Beads (catalog no. 37002D; Thermo Fisher Scientific) and SPRIselect beads (catalog no. B23318; Beckman Coulter). Purified cDNA was amplified as follows: 98°C for 3 min; 12 times (98°C for 15 s, 67°C for 20 s, 72°C for 60 s); 72°C for 60 s; hold 4°C. Amplified cDNA was purified using SPRIselect beads and sheared to ∼200 bp with a Covaris S2 instrument using the manufacturer’s recommended parameters. Sequencing libraries were generated with unique sample indices for each sample. Libraries for samples 1–3 and 4–5 were multiplexed respectively and sequenced on an Illumina NextSeq 500 (NextSeq control software v2.0.2/ Real Time Analysis v2.4.11) using a 150-cycle NextSeq 500/550 High Output Reagent Kit v2 (FC-404-2002; Illumina) in stand-alone mode as follows: 98 bp (read 1), 14 bp (I7 index), 8 bp (I5 index), and 485 10 bp (read 2).
8
Illumina RNA-Seq Library Preparation
9
Illumina NextSeq 500 Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Extracted DNA was employed to prepare 150-bp paired-end sequencing libraries using the Illumina Nextera XT Library Preparation Kit (Illumina Inc., San Diego, CA, USA). Sequencing was performed on the Illumina NextSeq 500 platform using a NextSeq 500/550 High Output Reagent kit v2 (300 cycles), in accordance with the standard Illumina sequencing protocols.
10
Nextera XT Whole-Metagenome Shotgun Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Whole-metagenome shotgun libraries were prepared using the Nextera XT kit in accordance with the Nextera XT DNA Library Preparation Guide from Illumina, with the exception that tagmentation time was increased to 7 min. Samples were sequenced on the Illumina NextSeq 500 in the Teagasc sequencing facility, with a NextSeq 500/550 High Output Reagent Kit v2 (300 cycles), in accordance with standard Illumina sequencing protocols.
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!