H. werneckii cells were grown to midexponential phase, and 200 ml of culture was used for nucleosome-bound gDNA preparation, following the protocol of Tsui et al. (2012) (link) but omitting the gel extraction step as described by Henikoff et al. (2011) (link). DNA was digested with titrations (12.5–50 U) of micrococcal nuclease (Fermentas), selecting for library preparation samples that showed >80% mononucleosomes based on agarose gel analysis. Two independent DNA samples were converted to sequencing libraries using NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs), selecting for 150 bp inserts with Agencourt AMPure XP Beads (Beckman Coulter). Libraries were sequenced on a HiSequation 2500 (Illumina), generating paired 100 base reads.
Hisequation 2500
Manufactured by Illumina
Sourced in Switzerland
The HiSequation 2500 is a high-throughput DNA sequencing system designed for large-scale genomic studies. It utilizes advanced sequencing-by-synthesis technology to generate high-quality sequencing data. The system is capable of processing multiple samples simultaneously, making it suitable for a wide range of genomic applications.
Automatically generated - may contain errors
Lab products found in correlation
Agencourt ampure xp bead, by Beckman Coulter (1 mentions)
Micrococcal nuclease, by Thermo Fisher Scientific (1 mentions)
Prepease dna isolation kit, by Thermo Fisher Scientific (1 mentions)
Truseq stranded rna lt guide, by Illumina (1 mentions)
Hincii, by New England Biolabs (1 mentions)
Truseq adapter, by Illumina (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Truseq dna pcr free lt library preparation protocol, by Illumina (1 mentions)
Nanodrop nd 8000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Qubit 1.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Ampure xp, by Beckman Coulter (1 mentions)
Dneasy blood and tissue dna kit, by Qiagen (1 mentions)
High fidelity sbfi, by New England Biolabs (1 mentions)
Tri reagent, by Thermo Fisher Scientific (1 mentions)
Truseq srna kit, by Illumina (1 mentions)
Masterpure yeast rna purification kit, by Illumina (1 mentions)
Truseq stranded mrna kit, by Illumina (1 mentions)
Truseq nano dna kit, by Illumina (1 mentions)
Masterpure yeast dna purification kit, by Illumina (1 mentions)
Truseq dna v2, by Illumina (1 mentions)
25 protocols using hisequation 2500
1
Nucleosome-bound DNA Sequencing in H. werneckii
2
Whole Genome Sequencing of Yeast
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Diploid colonies or spore colonies from tetrads were independently cultured overnight at 30° in YPD liquid medium. Genomic DNA was extracted from each culture using the PrepEase DNA isolation kit from Affymetrix following the manufacturer’s protocol. Whole genome sequencing was performed on Illumina HiSequation 2500 machines at Fasteris SA, Switzerland.
3
Comprehensive Maize Transcriptome Profiling
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The library for RNA sequencing was prepared based on Illumina standard instruction (TruSeq Stranded RNA LT Guide). According to the instructions in the HiSequation 2500 user guide, the library DNA was checked for its concentration and size distribution to ensure it met the request of Illumina HiSequation 2500 system before sequencing was performed. Evaluation of reads’ quality was accomplished using FastQC [60 ]. Any reads were removed if they were less than 40 bp using Btrim (set the parameter –l = 40) [61 (link)], and remaining reads were used for further analysis. cDNA sequence file (downloaded from www.maizegdb.org ) derived from Maize B73 genome assembly (V4) [52 (link)] was used as a reference. The Salmon software (version: 1.1.0) was used for reads mapping to the reference cDNA sequences and calculating the transcript per million (TPM) mapped reads of each transcript using quasi-mapping method [62 (link)]. The P-value of differential expression was calculated in the R environment (version 3.6.3, https://www.r-project.org/ ) using EdgeR package [63 (link)] (version: 3.28.1), for it has well performance in the identification of DEGs using three biological replicates [64 (link)]. EdgeR was downloaded from the Bioconductor website (www.bioconductor.org ). GO enrichment analysis was performed using agriGO (version:2.0) [65 ].
4
Plasma-Based Genomic Profiling of Cancer
5
GBS Library Construction for Tetrasomic Polyploids
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GBS libraries were prepared following Heffelfinger et al. (2014) (link). The methylation-sensitive restriction enzyme HincII [R0103; New England Biolabs (NEB)], which recognizes a degenerate six-bp sequence, was used for digestion. Libraries were constructed for the 169 F1 progenies and the two parents and were sequenced as 75-bp paired-end reads on the Illumina HiSequation 2500 in rapid run mode by the Yale Center for Genome Analysis (http://medicine.yale.edu/keck/ycga/index.aspx ) following the manufacturer’s protocol. Depths of coverage for each sample are provided in Supplemental Material, Table S1 . De novo SNP discovery and genotype calling was conducted using the Tassel 3.0 Universal Network Enabled Analysis Kit (UNEAK) pipeline (Lu et al. 2013 (link)). A greater number of reads are required to make accurate genotypic calls in tetrasomic polyploid populations than diploid populations. Thus, strict genotype calling thresholds were employed following the recommendations of Li et al. (2014) (link) in order to reliably distinguish between homozygotes (AAAA) and triplex heterozygotes (AAAB).
6
Illumina DNA Library Preparation
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DNA was sonicated to produce a 300–400 bp distribution, and then end-repaired and A-tailed enzymatically using Illumina’s standard TruSeq DNA library preparation. These products were ligated to Illumina Truseq adapters. Libraries, following indexing via PCR, were size selected to remove adapter and PCR dimers, pooled, and cosequenced on two lanes of the Illumina HiSequation 2500 using a 2 × 100 base pair format. The unique sequences incorporated during library construction were subsequently used to identify the source of each read, using postsequencing bioinformatics.
7
Comprehensive miRNA and mRNA Sequencing
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We generated miRNA sequencing data from 1,303 primary, 22 refractory, and 37 relapse samples, as well as mRNA sequencing data from 127 primary and 37 relapse samples (Fig 1A and Data Supplement). Sequencing for all samples was performed by using the Illumina Hi-SEquation 2500 (Illumina, San Diego, CA). miRNA sequencing data were aligned to hg19 and annotated on the basis of miRBase (version 21).19 (link) All data sets and sequences are available online (dbGaP accession no.: phs000465; SRA accession no.: SRP012000).20 ,21
8
RNA Isolation from H. werneckii Cells
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H. werneckii cells were grown to midexponential phase, and 50 ml of culture was pelleted, flash frozen in liquid nitrogen, and stored at −80° until RNA isolation. Pelleted cells were ground in a mortar and pestle maintained in a liquid nitrogen bath and the grindate used for total RNA isolation using TRIzol Reagent according to the manufacturer’s instructions (Thermo Fisher Scientific). Two independent RNA samples were converted to sequencing libraries using Illumina’s TruSeq RNA version 2 Library Preparation Kit, and sequenced on a HiSequation 2500 or MiSeq (Illumina) to generate paired-end 100 base reads.
9
High-Throughput RNA-Seq Analysis Pipeline
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Using 2 mg of RNA per sample, sequencing libraries were generated using NEBNext UltraTM RNA Library Prep Kit for Illumina (New England Biolabs, Ipswich, MA, USA). cDNA libraries were sequenced on Illumina HiSEquation 2500 yielding ∼15 million 50 bp single-end reads per sample. Overall quality was assessed with FastQC v. 0.11.5, low-quality bases were trimmed off with Trimmomatic v. 0.33 [50 (link)], followed by alignment to the Mus musculus genome draft GRCm38.84 using STAR v. 2.5.0 [51 (link)]. For visualization and hierarchical clustering, reads were normalized using the transcripts per million method [52 (link)], but raw read counts were used for differential expression analysis using DESeq2 v. 1.14 [53 (link)].
10
Illumina TruSeq DNA Sequencing Protocol
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DNA was processed using the Illumina TruSeq DNA PCR-Free LT Library Preparation protocol, to make single indexed, 550 bp insert libraries. These were titred individually using an Applied Biosystems Step One kit with Kapa Biosystems Illumina Library Quantification Kit reagents. After flowcell clustering using a TruSeq PE Cluster Kitv3-cBot-HS kit, sequence data were generated as paired, 101 base reads by an Illumina HiSequation 2500 using SBS v3-HS reagents. “Stage 1” pools of 24 samples with compatible indices were created and a single lane of each pool was sequenced. Based on demultiplexing results from the initial lane of sequence, the library titres were refined and a new “stage 2” pool created to correct for excesses/deficiencies in numbers of reads per library from the first lane. Two lanes of the stage 2 pool sequence were then run. Reads from all three runs were merged for each library.
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