DVP samples were processed on a liquid handling platform (Bravo, Agilent Technologies). Extracted cells were collected at the bottom of each well by acetonitrile‐supported centrifugation and vacuum evaporation until dryness. Thereafter, lysis was accomplished in 6 μl 70 mM TEAB, 0.013% DDM and incubation at 95°C for 60 min, complemented by additional 60 min at 75°C in 12% (v/v) acetonitrile (by adding 1 μl 80% ACN). Proteins were digested using 4 ng LysC and 6 ng trypsin overnight at 37°C and labeled as described above. For the reference channel, a consecutive tissue slide – containing mainly but not exclusively tumor material – was collected from a PEN membrane slide into a 96‐well plate (Covaris) using a scalpel and processed analogous to the DVP samples using 42 μl of lysis buffer and additional sonification (LE220‐plus: peak Power: 450.0, duty factor: 50%, cycles: 200, average power: 225, Covaris). The following steps of digestion, purification and labeling were performed as described above in sample prepartion of the reference channel. Evotip loading was accomplished as described above using 25 ng of the bulk tissue as reference channel (∆0) combined with the target channels. Two replicates of each melanoma type were measured as target ∆4 and ∆8, respectively.
Le220 plus
Manufactured by Covaris
The LE220-plus is a high-performance laboratory instrument designed for sample preparation. It utilizes advanced acoustic energy technology to process samples in a controlled and efficient manner. The LE220-plus is capable of performing various sample preparation tasks, including homogenization, cell lysis, and DNA/RNA extraction. The device's core function is to provide a versatile and reliable solution for researchers and scientists working in diverse laboratory settings.
Automatically generated - may contain errors
Lab products found in correlation
Kapa hyper prep kit, by Roche (2 mentions)
Hiseq instrument, by Illumina (2 mentions)
Human gdna, by Promega (2 mentions)
Sureselectxt target enrichment system for paired end multiplexed sequencing library, by Illumina (2 mentions)
Iseq 100, by Illumina (1 mentions)
Novaseq 6000 platform, by Illumina (1 mentions)
Truseq dna pcr free library prep protocol, by Illumina (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Truseq dna pcr free ht sample preparation kit, by Illumina (1 mentions)
Smarter ultra low input rna kit for sequencing v4, by Takara Bio (1 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Hiseq sr cluster kit v4, by Illumina (1 mentions)
Trizol, by Zymo Research (1 mentions)
8 microtube strip v1, by Covaris (1 mentions)
Suredesign, by Agilent Technologies (1 mentions)
Kapa library quantification kit, by Illumina (1 mentions)
Agilent 2100 bioanalyser, by Agilent Technologies (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Dna 7500 assay, by Agilent Technologies (1 mentions)
9 protocols using le220 plus
1
Quantitative Proteomics of Melanoma
2
Whole-Genome Sequencing of GIAB Samples
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We ordered the GIAB samples from the Coriell Institute (NA24385, NIST ID HG002; NA24149, NIST-ID HG003 and NA24143, NIST-ID HG004). DNA concentration was measured by Qubit.
The library was constructed according to Illumina TruSeq DNA PCR Free Library Prep protocol HT (Illumina Inc., San Diego, CA, USA) for whole genome sequencing. Briefly, the protocol steps were: (1) fragmentation of 1 μg genomic DNA to 350 bp inserts by Covaris LE220-plus, (2) cleanup of fragmented DNA, (3) repair ends, (4) removal of large and small DNA fragments, (5) 3′-end adenylation and (6) adapter ligation. The resulting library was quantified and quality-assessed with the iSeq100 (Illumina). The GIAB samples were sequenced with the NovaSeq 6000 platform (Illumina) using S4 flow cells with 300 cycles (2 × 150 reads) and measured 2 × to reach an average coverage of 35×.
The library was constructed according to Illumina TruSeq DNA PCR Free Library Prep protocol HT (Illumina Inc., San Diego, CA, USA) for whole genome sequencing. Briefly, the protocol steps were: (1) fragmentation of 1 μg genomic DNA to 350 bp inserts by Covaris LE220-plus, (2) cleanup of fragmented DNA, (3) repair ends, (4) removal of large and small DNA fragments, (5) 3′-end adenylation and (6) adapter ligation. The resulting library was quantified and quality-assessed with the iSeq100 (Illumina). The GIAB samples were sequenced with the NovaSeq 6000 platform (Illumina) using S4 flow cells with 300 cycles (2 × 150 reads) and measured 2 × to reach an average coverage of 35×.
3
High-Coverage Sequencing of Parental Genomes
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PCR-free Illumina libraries were generated from 1 μg genomic DNA using a Covaris LE220-plus to shear the DNA and the TruSeq® DNA PCR-Free HT Sample Preparation Kit (Illumina) for library generation. The median insert sizes were approximately 400 bp. Libraries were tagged with unique dual index DNA barcodes to allow pooling of libraries and minimize the impact of barcode hopping. Libraries were pooled for sequencing on the NovaSeq 6000 (Illumina) to obtain at least 750 million 151-base read pairs per library. This resulted in 49.3X coverage of the parental genomes.
4
Genomic DNA Shearing and Library Prep
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Genomic DNA from each organism was first sheared using ultrasonic shearing (Covaris LE220-plus) using the following settings: peak power = 450W, duty factor = 30%, cycles/burst = 200. DNA was sheared to an average size of 75 bp in Tris-HCl buffer (pH=8) and applied in multiple cycles of 30 minutes each for a total of 60-90 minutes, allowing time for the water bath to cool between cycles such that the maximum temperature of the samples did not exceed 15°C. Fragment size can be adjusted to meet the desired resolution (see Figure S12). After shearing, up to 1 μg of each genomic DNA sample was used to prepare fragment libraries using the KAPA HyperPrep kit and standard manufacturer's protocol. During the adapter ligation step, custom annealed Y-adapters were introduced at a concentration of 15pM (5 μL adapters in a reaction volume of 110 μL, final adapter concentration = 0.7 pM). These custom adapters were prepared by annealing a full-length i5 index adapter with an index-less stub i7 adapter. Ligated libraries were amplified for 8-10 cycles using primers P1 and P2stub. Strains used in this work and oligonucleotide and barcodes are detailed in the Table S3 and Table S5.
For experiments using Arabidopsis thaliana Col-0, the gDNA libraries were constructed from gDNA sheared to an average fragment size of 150 bp.
For experiments using Arabidopsis thaliana Col-0, the gDNA libraries were constructed from gDNA sheared to an average fragment size of 150 bp.
5
RNA-Seq Library Preparation Pipeline
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RNA samples were extracted with TRIZOL (500 μL, Zymo Research). RNA sequencing libraries were prepared by using SMARTer Ultra Low Input RNA Kit for Sequencing v4 (TaKaRa Clontech) and KAPA Hyper Prep Kit (KAPA Biosystems) according to the manufacturer's protocol. The resulting double stranded cDNA was sheared using a Covaris LE220 Plus (Covaris) with a 200 bp peak in the 50μl volume setting. The fragmented cDNA underwent end repair, 3′ end adenylation and ligation with barcoded adapters. The libraries were validated using the Agilent Bioanalzyer DNA High Sensitivity kit (Agilent), and quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific). The sequencing library templates were prepared for sequencing using the Illumina HiSeq SR Cluster V4 Kit. Sequencing runs were performed on an Illumina HiSeq 2500 using the single read mode of 51 cycles of read 1 and 7 cycles of index read with the SBS V4 Kit. Real-time analysis (RTA) 2.2.38 software was used to process the image analysis and base calling.
6
RNA Bait Capture of Chlamydia trachomatis
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We used a methodology for RNA bait capture of C. trachomatis described in detail by Bowden et al. (2021) (link). Human gDNA (Promega, San Luis Obispo, CA) was added to the extracted gDNA from the clinical swabs to reach a total input of 3 μg/130 μL for fragmentation and library prep. Samples were sheared on the Covaris LE220 plus (Covaris, Woburn, MA). After shearing and magnetic bead purification, the SureSelectXT Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library (VC2 Dec 2018) and all recommended quality control steps were performed on all gDNA samples. The 2.698 Mbp RNA bait library consisted of 34,795 120-mer probes spanning 85 GenBank C. trachomatis reference genomes (Bowden et al., 2021 (link))(Agilent Technologies, INC, Santa Clara, CA, reference: ELID: 3173001). A 16-hour incubation at 65°C was performed for RNA bait library hybridization. Post-capture PCR cycling was set at 12 cycles based on a capture library size > 1.5 Mb. The libraries were paired end sequenced for 150 nt using an Illumina HiSeq instrument. Sequence data from this project was submitted to the NCBI Sequence Read Archive under the BioProject accession ID: PRJNA609714 .
7
C. trachomatis Genome Enrichment Protocol
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Samples were quantified using the Qubit® 2.0 Fluorometer, and human gDNA (Promega, San Luis Obispo, CA) was added to reach a total input of 3 µg/130uL for fragmentation and library prep.
Samples were sheared on the Covaris® LE220plus using the 8 microTUBE strip V1 (PM# 520053; Covaris, Woburn, MA) with the base pair (bp) mode set to 250-300 bp following the manufacturer's instructions.
RNA Bait library design A 2.698 Mbp RNA bait library consisting of 34,795 120-mer probes spanning 85 GenBank C. trachomatis reference genomes were designed using Agilent SureDesign. No plasmid probes were included in the RNA bait library construction. The bait library was synthesized by Agilent Technologies.
The custom designed RNA bait library (ELID: 3173001) used in this study can be retrieved by contacting Agilent Technologies, INC (Santa Clara, CA).
Samples were sheared on the Covaris® LE220plus using the 8 microTUBE strip V1 (PM# 520053; Covaris, Woburn, MA) with the base pair (bp) mode set to 250-300 bp following the manufacturer's instructions.
RNA Bait library design A 2.698 Mbp RNA bait library consisting of 34,795 120-mer probes spanning 85 GenBank C. trachomatis reference genomes were designed using Agilent SureDesign. No plasmid probes were included in the RNA bait library construction. The bait library was synthesized by Agilent Technologies.
The custom designed RNA bait library (ELID: 3173001) used in this study can be retrieved by contacting Agilent Technologies, INC (Santa Clara, CA).
8
RNA Bait Capture of Chlamydia trachomatis
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We used a methodology for RNA bait capture of C. trachomatis described in detail by Bowden et al(19 ). Human gDNA (Promega, San Luis Obispo, CA) was added to the extracted gDNA from the clinical swabs to reach a total input of 3 μg/130uL for fragmentation and library prep. Samples were sheared on the Covaris LE220 plus (Covaris, Woburn, MA). After shearing and magnetic bead purification, the SureSelectXT Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Library (VC2 Dec 2018) and all recommended quality control steps were performed on all gDNA samples. The 2.698 Mbp RNA bait library consisted of 34,795 120-mer probes spanning 85 GenBank C. trachomatis reference genomes(19 )(Agilent Technologies, INC, Santa Clara, CA, reference: ELID: 3173001). A 16-hour incubation at 65°C was performed for RNA bait library hybridization. Post-capture PCR cycling was set at 12 cycles based on a capture library size > 1.5 Mb. The libraries were paired end sequenced for 150 nt using an Illumina HiSeq instrument. Sequence data from this project was submitted to the NCBI Sequence Read Archive under the BioProject accession ID: PRJNA609714
9
Whole Genome Sequencing Library Prep
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The short-insert paired-end libraries for whole genome sequencing (WGS) were prepared from DNA of the same female used for long-read sequencing with a PCR-free protocol using KAPA HyperPrep kit (Roche) with some modifications. In short, 1.0 μg of genomic DNA was sheared on a Covaris LE220-Plus (Covaris) and sizeselected for the fragment size of 220-550 bp with AMPure XP beads (Agencourt, Beckman Coulter). The genomic DNA fragments were then end-repaired and adenylated. Next, compatible adaptors for Illumina platforms with unique dual indexes including unique molecular identifiers (Integrated DNA Technologies) were ligated. The libraries were quality controlled on an Agilent 2100 Bioanalyser with the DNA 7500 assay (Agilent) for size and quantified by Kapa Library Quantification Kit for Illumina platforms (Roche).
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