Total bacterial DNA was extracted from the colonic digesta using a bead-beating and phenol-chloroform method as previously described by Zoetendal et al. (13 (link)). DNA concentration was measured by a Nano-Drop 1000 spectrophotometer (Thermo Scientific Inc, Wilmington, DE, USA). The extracted DNA samples were sent to Biozeron Biotechnology Co., Ltd (Shanghai, China) for 16s rRNA gene sequencing. The V3-V4 regions of the bacterial 16S rRNA gene were amplified by PCR using universal primers 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′). The Qubit 3.0 fluorometer was used to quantify each PCR product for uniform mixing. The Illumina Truseq DNA Library Construction Kit was used for the library construction of mixed samples. Sequencing was conducted on an Illumina platform.
Truseq dna library construction kit
Manufactured by Illumina
Sourced in United States
The TruSeq DNA Library Construction Kit is a laboratory equipment product designed for preparing DNA samples for sequencing. It provides the necessary reagents and protocols to construct DNA libraries from a variety of sample types, enabling researchers to prepare samples for downstream analysis.
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Lab products found in correlation
2 protocols using truseq dna library construction kit
1
Colonic Microbiome DNA Extraction and Sequencing
2
Illumina HiSeq Xten Library Preparation
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The present study was performed by Beijing Novogene Bioinformatics Technology Co., Ltd, Beijing, China (www.novogene.com/ ). Briefly, the qualified DNA samples were randomly sheared into DNA fragment sizes of 350 bp using the Covaris S220 Focused UItrasonicator (Covaris, Woburn, MA, USA), and library construction was performed according to the manufacturer's protocol of the TruSeqDNA Library Construction kit (Illumina, Inc., San Diego, CA, USA). The Qubit® 2.0 Fluorometer was used first for preliminary quantification following the completion of library construction, following which the Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Waldbronn, Germany) was used for determining the insert size of the library. This was followed by accurate quantification using the quantitative polymerase chain reaction method to ensure the library effective concentration was >2 nM. Sequencing was performed using the Illumina HiSeq Xten platform (Illumina, Inc.) to obtain the raw reads. Reads with adapter sequences or of low quality were filtered out to obtain clean reads, which were used in the following analysis.
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