DNA was isolated from cultured satellite cells using OMEGA TISSUE DNA Kit (2000) (n = 3 for each time point). The concentration and quality of DNA were assessed by NanoDrop® ND-1000 and agarose gel electrophoresis. The library was prepared according to MeDIP library development flow. Sequencing was performed by SHANGHAI BIOTECHNOLOGY CORPORATION. The amount of DNA used for sequencing was about 2 μg. The sequencing process included the following: (1) Library construction was performed and the integrity was verified with Agilent 2100 (Agilent Technologies, Santa Clara, CA, United States). (2) Cluster generation and primer hybridization were completed on the cBot equipped with the Illumina HiSeq sequencing instrument according to the corresponding process shown in the cBot User Guide. (3) The sequencing process was controlled by data collection software provided by Illumina, and real-time data analysis was carried out.
Hiseq sequencing instrument
Manufactured by Illumina
Sourced in United States
The HiSeq sequencing instrument is a high-throughput DNA sequencing platform developed by Illumina. It is designed to perform next-generation sequencing, enabling rapid and large-scale DNA analysis. The core function of the HiSeq is to generate DNA sequence data from biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Agilent 2100, by Agilent Technologies (1 mentions)
Ribo zero rrna removal kit, by Illumina (1 mentions)
Truseq stranded total rna library prep kit, by Illumina (1 mentions)
Flow cell, by Illumina (1 mentions)
Bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Truseq small rna library preparation kit, by Illumina (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
5 protocols using hiseq sequencing instrument
1
Satellite Cell DNA Methylation Profiling
2
RNA Sequencing Library Preparation
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Library preparation of RNA and high-throughput sequencing were carried out by Cloud-Seq Biotech (Shanghai, China). According to the supplier’s instructions, the Ribozero rRNA Removal Kit (Illumina, San Diego, CA, USA) was applied to remove the rRNAs from total RNA. RNA preprocessing for constructing the sequencing library was implemented with the TruSeq Stranded Total RNA Library Prep Kit (Illumina). Quality control and quantification of the libraries were performed with the Bio-Analyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). The 10 pM libraries were changed into single-stranded DNA molecules, captured on Illumina Flow Cells, and then amplified in situ as clusters, which were subsequently sequenced for 150 cycles on the Illumina Hi-Seq sequencing instrument, using the two-terminal mode (PE mode).
3
Embryonic RNA Sequencing Protocol
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All collected embryos were pooled, then soaked in TRIzol reagent (Invitrogen) and subjected to 10 rounds of flash freeze–thaw cycles in liquid nitrogen to thoroughly disrupt the eggs. Released total RNAs were extracted according to the manufacturer's instructions. mRNA purification and sequencing were performed by Novogene using the Illumina TruSeq stranded mRNA library preparation method (paired-end, 150 bp) on a HiSeq sequencing instrument (Illumina). For small RNA sequencing, total RNAs were first treated with RNA 5′-polyphosphatase (Lucigen) to convert all of the 5′-triphosphates to monophosphates before purified again by TRIzol reagents. Small RNA extraction and the preparation of sequencing libraries were also performed by Novogene using the Illumina TruSeq small RNA library preparation kit.
4
Whole-genome sequencing of chicken alleles
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DNA was prepared from blood samples of single individuals representing the D*V (White Crested Black Polish, USA), D*C (Sicilian Buttercup, France) and D*N (Single Comb Dark Brown Leghorn, USA) alleles. The DNA was used to construct paired-end libraries with average insert size of approximately 220 bp and these libraries were subjected to whole genome sequencing using a HiSeq sequencing instrument (Illumina). Sequencing reads (2 x 100bp) were aligned to the chicken reference genome (galgal3) using the Burrows Wheeler Aligner (BWA) [35 (link)], revealing average depths of coverage of 24, 25 and 34 for D*V, D*N and D*C, respectively. The aligned reads were subjected to duplicate flagging using Picard Tools (http://picard.sourceforge.net ) and to SNP calling using the Genome Analysis Toolkit (GATK) Unified Genotyper version 2.4.9 [36 (link)]. Identified raw SNPs were filtered based on GATK best practice variant detection and genotypes with a PHRED genotype quality ≥ 20 were used in subsequent steps. SNP- and genotype calls were compared to SNPs detected in DNA pools from wild- and domestic chickens in a previously published study [13 (link)].
5
Genomic Analysis of Ceftolozane-Tazobactam Resistant Strains
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Strains with ceftolozane-tazobactam MIC values ≥ 8 μg/mL were examined.
Genomic DNA was extracted using Qiagen DNeasy UltraClean kits (Qiagen, Valencia, CA, USA) and quantified using the Nanodrop™ ND-1000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA).
For whole-genome sequencing, DNA sequences were obtained on the Illumina HiSeq sequencing instrument (Illumina, San Diego, CA, USA) with 2 × 150 bp pair-end reads with a target coverage depth of approximately 150×. All analyses were carried out using Qiagen's CLCBioGenomics Workbench version 11.
For β-lactamase resistance, genes were identified by Illumina whole-genome sequencing. β-lactamase gene inclusion was 72% and 80% for minimum nucleotide sequence identity and minimum sequence length, respectively.
For porin gene identification, ompk35 and ompK36 in K. pneumoniae and oprD in P. aeruginosa were searched by TBLASTN; for ftsI (encoding PBP3) gene analysis, searching was on a species-specific basis in de novo assemblies of each genome.
The appropriate multilocus sequence typing scheme and allelic profile of each of the guided assemblies was determined computationally (using the ‘find best match using k-mer spectra’ tool in CLC genomics).
Genomic DNA was extracted using Qiagen DNeasy UltraClean kits (Qiagen, Valencia, CA, USA) and quantified using the Nanodrop™ ND-1000 spectrophotometer (ThermoFisher Scientific, Waltham, MA, USA).
For whole-genome sequencing, DNA sequences were obtained on the Illumina HiSeq sequencing instrument (Illumina, San Diego, CA, USA) with 2 × 150 bp pair-end reads with a target coverage depth of approximately 150×. All analyses were carried out using Qiagen's CLCBio
For β-lactamase resistance, genes were identified by Illumina whole-genome sequencing. β-lactamase gene inclusion was 72% and 80% for minimum nucleotide sequence identity and minimum sequence length, respectively.
For porin gene identification, ompk35 and ompK36 in K. pneumoniae and oprD in P. aeruginosa were searched by TBLASTN; for ftsI (encoding PBP3) gene analysis, searching was on a species-specific basis in de novo assemblies of each genome.
The appropriate multilocus sequence typing scheme and allelic profile of each of the guided assemblies was determined computationally (using the ‘find best match using k-mer spectra’ tool in CLC genomics).
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