Focus: Ruben et. al. from Boston University School of Medicine developed a ChIP-Seq sample preparation protocol for whole body C. elegans extracts. The authors of this application note provide a step-by-step sample preparation workflow and highlight the key advantages of using the Covaris S220 Focused-ultrasonicator.
This document provides users with a complete set of protocols that encompass all Covaris Adaptive Focused Acoustics™
(AFA) instruments for DNA shearing to 150 to 200 bp with 50 to 55 µl of sample volume, to be readily compatible with the Agilent SureSelect Target Enrichment System (XT or XT2). The goal is to provide the most precise, pre-validated protocols to ensure the best outcome and compatibility for library preparation and subsequent sequencing applications.
Next-Generation Sequencing (NGS) platforms require controlled generation of DNA fragments to produce high-quality sequencing data. Covaris leverages its proprietary and patented Adaptive Focused Acoustics (AFA™) technology to convert focused high frequency acoustic energy into mechanical force to randomly fragment nucleic acids in an isothermal and non-contact environment.
Controlled generation of DNA fragments is a critical step required by all Next-Generation Sequencing (NGS) platforms. Covaris truSHEAR employs Adaptive Focused Acoustics™ (AFA) technology for the controlled mechanical shearing of the phosphodiester backbone of nucleic acids. AFA hydrodynamic shear force-based fragmentation is purely mechanical and designed to be isothermal, ensuring both unbiased fragmentation and high recovery of double-stranded and single–stranded DNA.
These protocols have been optimized for both mammalian cell lines and fly embryos and reliably fragment chromatin in low volumes (from 20 to 50 μl) from down to 10,000 mammalian cells and 5 stage-17 Drosophila embryos. They provide a good starting point when aiming to develop and optimize reduced cell and volume chromatin shearing protocols for ChIP experiments.
Dried Blood Spots (DBS) provide an easy and inexpensive way to collect and store peripheral blood specimens from infants, children nd adults. The use of DBS allows for less invasive procedures for patients and easier shipment while still providing the ability to run molecular or clinical biochemical assays. This convenient method for the long term room-temperature storage of materials also minimizes storage and archival space. The truXTRAC DBS DNA kit is designed for controlled and efficient extraction of next-generation sequencing (NGS)-grade DNA from DBS samples using Adaptive Focused Acoustics® (AFA®). Covaris AFA enables sample rehydration while providing simultaneous cell lysis and controlled mechanical DNA shearing, resulting in high-yield, high-quality and NGS library preparation ready DNA.
Mycobacterial identification using MALDI-ToF MS (MALDI) has been hindered by inadequate extraction methods. Adaptive Focused Acoustics™ uses concentrated ultrasonic energy to achieve cellular disruption. Using this technology, we developed a rapid mycobacterial inactivation/protein extraction method for MALDI- based identification. Agreement for identification to the species level versus conventional identification was stratified by log confidence cut-offs of ≥2.0, ≥ 1.8, or ≥1.7. A total of 182mycobacterial isolates were tested. Complete inactivation of all species/strains was achieved after 2 min. Using a log confidence cut-off of ≥2.0, overall
agreement for the commercial method (CM) was 41.7% versus 66.7% for the novel method (NM). For the CM, agreement increased to 66.7% and 83.3% using log confidence cut-offs of ≥1.8 and ≥1.7, respectively; for the NM, agreement was 100% for both cut-offs with all isolates. With no alteration to the existing database, overall
agreement for the NM was 83.4%, largely due to low scores for clinical isolates of M. chelonae and M.mucogenicum. Addition of spectra froma single clinical strain of each species to the existing database increased overall agreement to 93.1%.
Covaris provides tools and technologies to improve pre-analytical sample preparation, enable novel drug formulations, and manage compounds in the drug discovery process.