Single cell suspensions were diluted to 280 cells/ul and processed using the Drop-seq platform as previously published (Macosko et al., 2015 (link), Green et al., 2018 (link)). Barcoded microparticle beads (MACOSKO-2011–10, Lots 090316, 072817, and 060718, ChemGenes Corporation) were used. Briefly, cells, barcoded microparticle beads and lysis buffer were co-flown into a microfluidic device and captured in nanoliter-sized droplets. After droplet collection and breakage, the beads were washed, and cDNA synthesis occurred on the bead using Maxima H-minus RT (Thermo Fisher Scientific) and a Template Switch Oligo. Excess oligos were removed by exonuclease I digestion. cDNA amplification was done for 15 cycles from pools of 2,000 beads using HotStart ReadyMix (Kapa Biosystems) and the SMART PCR primer. Individual PCRs were purified and pooled for library generation. A total of 600 pg of amplified cDNA was used for a Nextera XT library preparation (Illumina) with the New-P5-SMART PCR hybrid oligo, and a modified P7 Nextera oligo with 10 bp barcodes. Sequencing was performed on a HiSeq-2500 (Illumina) for read length of 112 nt or 115 nt, with the Read1CustomSeqB primer. Oligo sequences are the same as previously described (Macosko et al., 2015 (link), Green et al., 2018 (link)).
Nextera xt library preparation
Manufactured by Illumina
Sourced in United States
The Nextera XT library preparation kit is a DNA library preparation solution for next-generation sequencing. It enables the preparation of DNA libraries from small input amounts, facilitating the streamlined generation of sequencing-ready libraries.
Automatically generated - may contain errors
Lab products found in correlation
Miseq platform, by Illumina (5 mentions)
Hotstart readymix, by Roche (4 mentions)
Maxima h minus rt, by Thermo Fisher Scientific (4 mentions)
Nextseq 550, by Illumina (3 mentions)
Macosko 2011 10, by Chemgenes (3 mentions)
Dnase 1, by Thermo Fisher Scientific (3 mentions)
Rnase 1, by Thermo Fisher Scientific (3 mentions)
Hiseq 2500, by Illumina (2 mentions)
Ampure bead, by Beckman Coulter (2 mentions)
Qubit broad range assay, by Thermo Fisher Scientific (2 mentions)
Sorbitol, by Merck Group (1 mentions)
Dneasy blood tissue kit, by Qiagen (1 mentions)
Lyticase, by Merck Group (1 mentions)
High sensitivity d1000 screentape assay, by Agilent Technologies (1 mentions)
Qubit high sensitivity assay, by Thermo Fisher Scientific (1 mentions)
Qiaamp fast dna stool mini kit, by Qiagen (1 mentions)
Agilent 4200 tapestation, by Agilent Technologies (1 mentions)
Superscript 3 kit, by Thermo Fisher Scientific (1 mentions)
Nucleospin soil kit, by Macherey-Nagel (1 mentions)
Qubit dsdna high sensitivity assay, by Thermo Fisher Scientific (1 mentions)
21 protocols using nextera xt library preparation
1
Single-cell transcriptome profiling using Drop-seq
2
Genomic DNA extraction and sequencing of Candida and Enterobacter
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Three milliliters of ON C. parapsilosis cultures (37 °C with aeration in Sab-chl broth) were harvested by centrifugation (10 min, 5000× g), re-suspended in 600 µL of lysis buffer (1 M sorbitol, 100 mM EDTA, 14 mM β-mercaptoethanol, 200 U of lyticase, all from Sigma, St. Louis, MO, USA) and incubated at 30 °C for 30 min. The sample was then centrifuged (10 min, 5000× g) with the pellet being processed according to the manufacturer’s instructions for the Qiagen DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany). For E. cloacae complex isolates, the same DNA extraction kit was used starting with one loop of ON cultures on MH agar. DNA was subjected to Nextera XT library preparation (Illumina, San Diego, CA, USA) prior to paired-end sequencing (2 × 250 bp or 2 × 150 bp) on either a MiSeq or a NextSeq 550 instrument (Illumina, San Diego, CA, USA), according to the manufacturer’s instructions.
3
Fecal DNA Extraction and Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The QIAamp Fast DNA Stool Mini Kit (Qiagen, Hilden, Germany) was used following the manufacturer’s protocol for the extraction of total DNA from fecal and luminol samples. Before extraction, the ZymobIOMICs spike-in control 1 (Zymo Research, Irvine, CA, United States) was added, 20 μL for every 20 mg of feces. Extracted DNA was measured using the Qubit High Sensitivity assay (Thermo Fisher Scientific) before standardization for paired-end Nextera XT library preparation (Illumina, San Diego, CA, United States). Library quality was inspected with the 4200 Agilent Tapestation (Agilent Technologies, Santa Clara, CA, United States) using the High Sensitivity D1000 ScreenTape assay (Agilent Technologies) before sequencing with an Illumina MiSeq platform (Illumina Inc., San Diego, CA, United States).
4
Viral Genome Reconstruction via NGS
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The samples that showed positive results in the ZDC assay were submitted to NGS (Next Generation Sequencing). At first, the first-strand cDNA synthesis was performed with random primers and SuperScript III kit (Life Technologies, Grand Island, NY, USA); the second strand of cDNA was produced using the Klenow FRAGMENT kit (New England Biolabs, Ipswich, MA, USA). After, the reverse transcription product was used to Nextera XT library preparation (Illumina, San Diego, CA, USA). The paired-end, 300 pb sequences generated by MiSeq were demultiplexed using Illumina software. Assemblers were used for the reconstruction of viral genomes, including SOAPdenovo2, Abyss, meta-Velvet and CAP3, Mira and SPADES programs. The full or partial genomes were then evaluated using Geneious R8 (Biomatters, San Francisco, CA, USA).
5
Shotgun Sequencing of Sediment Microbiome
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was extracted twice per sample, using 0.25 g sediment, soil, or biofilm, with NucleoSpin Soil kits (Machery-Nagel, Düren, Germany) then pooled. DNA quality was assessed with a 1% agarose gel and concentration was measured with Qubit dsDNA high-sensitivity assays (ThermoFisher, Waltham, USA). 1 ng of DNA from three replicates of selected sediment samples (Cool, Beta, Epsilon) was prepared for shotgun sequencing on one NextSeq550 (Illumina) run in High Output mode using NexteraXT library preparation (Illumina, San Diego, USA). Additional sampling and 16S rRNA gene sequencing are described in the Supplementary Methods.
6
Genomic Analysis of Evolved Bacterial Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ancestor V14 and evolved variants 14EV1 and 14EV2 obtained in the evolution experiment were sequenced using Illumina chemistry as described before (Koomen et al., 2021 (link)). Briefly, cells were pelleted and resuspended in 450 μL DNA/RNA Shield (Zymo Research) at 4°C until DNA extraction. The DNA was extracted by BaseClear (Leiden, the Netherlands) and paired-end 2 × 150 bp short-reads were generated using a Nextera XT library preparation (Illumina). A NovaSeq 6,000 system (Illumina) was used to generate paired-end reads. Raw reads were trimmed and de novo assembled using CLC Genomics Workbench v 10.0 (Qiagen, Hilden, Germany). SNIPPY 3.2 (Torsten, 2015 ), and Pilon using the “--changes” argument (Walker et al., 2014 (link)) were used for SNP analysis of evolved variants against the LO28 WT as reference.
7
Next-generation sequencing of ARTIC amplicons
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
ARTIC amplicons were cleaned using AMPure beads (Agencourt, Beckman Coulter, USA) and library were prepared using the Nextera XT library preparation and the Nextera indexing kits (Illumina, USA) [45 ]. The indexed library was then quantified with Qubit 3 (Thermoscietific, USA). The prepared library was sequenced on a Illumina Miseq using 300 cycle MiSeq Reagent Kit V2, and the data was de-multiplexed by MiSeq Reporter.
8
Single-cell transcriptome profiling using Drop-seq
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Drop-seq was performed on cells collected from various points of differentiation, where a single sample per timepoint was diluted to 280 cells/µl and processed as described previously98 (link). Briefly, cells, barcoded microparticle beads (MACOSKO-2011-10, Lots 113015B and 090316, ChemGenes Corporation), and lysis buffer were co-flown into a microfluidic device and captured in nanoliter-sized droplets. After droplet collection and breakage, the beads were washed, and cDNA synthesis occurred on the bead using Maxima H-minus RT (ThermoFisher Scientific) and the Template Switch Oligo. Excess oligos were removed by exonuclease I digestion. cDNA amplification was done for 15 cycles from pools of 2000 beads using Hot Start Ready Mix (Kapa Biosystems) and the SMART PCR primer. Individual PCRs were purified and pooled for library generation. A total of 600 pg of amplified cDNA was used for a NexteraXT library preparation (Illumina) with the New-P5-SMARTPCR hybrid oligo, and a modified P7 Nexteraoligo with 10 bp barcodes. Sequencing was performed on a NovaSeq (Illumina) for read 2 length of 94 nt with the Read1 Custom Seq primer. Oligosequences are the same as previously described13 (link),98 (link).
9
Single-cell RNA-seq Library Preparation
10
16S rRNA Gene Profiling of Biofilm Communities
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To characterise biofilm communities, 16S rRNA gene V4 sequences were PCR-amplified from 1 μl of DNA extract using the AccuPrime High Fidelity PCR kit (Invitrogen Catalog No 12346094) with the primer pair 515F (5′ AGCMGCCGCGGTAA 3′) and 806R (5′ GGACTACHVGGGTWTCTAAT ′3) containing Illumina MiSeq adaptors and single-end barcodes. PCR temperature cycles weres: 98 °C for 3 s, 33 cycles of: 98 °C for 20 s, 50 °C for 30 s, 72 °C for 90 s; then 72 °C for a final 10 min. Amplicons were pooled in equal quantities, cleaned with AMPure beads (Beckman Coulter) and paired-end sequenced on the MiSeq platform following Nextera XT library preparation (Illumina).
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!