ATAC-seq was performed using the TruePrep DNA Library Prep Kit V2 for Illumina (Nanjing Vazyme, China) according to the manufacturer's instructions. Briefly, cell pellets were resuspended in lysis buffer, pelleted and tagmented by using the enzyme and buffer provided in the TruePrep DNA Library Prep Kit V2 for Illumina. Trimomatic (v. 0.39) was used for raw reads to remove low quality reads and adaptor sequences. The resulting trimmed reads were aligned to mm10 by Bwa-mem (v. 0.7.17). Duplicate reads were marked and removed by Picard MarkDuplicates (v. 2.25.6). The reads whose mapping quality >20 were used for further analysis. The reads that were aligned to mitochondria were removed by Samtools (v. 1.7) and then converted to BigWig profiles by Deeptools (v. 3.5.1) for visualization in the Integrative Genomics Viewer (v. 2.9.2). Peaks were identified by using the Macs2 (v. 2.2.7.1) with cutoff P-value 5-e2 and nomodel option. R package Diffbind (v. 2.16.2) was used to detect significantly differential peaks (P-value < 0.05 and |fold change| > 1.5). Bioconductor package ChIPseeker (v. 1.24.0) and TxDb.Mmusculus.UCSC.mm10.knownGene (v. 3.10.0) were used to annotate the differential peaks.
Trueprep dna library prep kit v2
Manufactured by Illumina
Sourced in China, United States
The TruePrep DNA Library Prep Kit V2 is a laboratory equipment product designed for DNA library preparation. It provides a streamlined workflow for generating DNA libraries suitable for next-generation sequencing applications.
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Lab products found in correlation
Hiseq x ten, by Illumina (13 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (6 mentions)
Novaseq 6000, by Illumina (6 mentions)
Vahts dna clean beads, by Vazyme (5 mentions)
Hiseq x ten platform, by Illumina (5 mentions)
Xgen exome research panel, by Integrated DNA Technologies (4 mentions)
Hiseq machine, by Illumina (4 mentions)
Qiaamp dna mini kit, by Qiagen (4 mentions)
Hiseq 2500, by Illumina (4 mentions)
Ampure xp bead, by Beckman Coulter (3 mentions)
Single cell full length mrna amplification kit, by Vazyme (3 mentions)
Hiseq system, by Illumina (3 mentions)
Dna clean beads, by Illumina (3 mentions)
Agencourt ampure xp bead, by Beckman Coulter (2 mentions)
Novaseq 6000 platform, by Illumina (2 mentions)
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Dneasy powersoil kit, by Qiagen (2 mentions)
Nextera xt dna library preparation kit, by Illumina (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Novaseq platform, by Illumina (2 mentions)
90 protocols using trueprep dna library prep kit v2
1
ATAC-Seq library preparation and analysis
2
ATAC-seq Library Preparation Protocol
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The ATAC-seq procedure was carried out following the previously published protocols [15 (link), 16 (link)] and TruePrep DNA Library Prep Kit V2 for Illumina. Briefly, 50,000 cells were washed with 50 ml of cold PBS and suspended in 50 ml of lysis buffer containing 10 mM Tris–HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, and 0.2% (v/v) IGEPAL CA-630. The cell suspension was centrifuged at 500 g for 10 min at 4 °C, followed by the addition of 50 ml of transposition reaction mix from the TruePrep DNA Library Prep Kit V2 for Illumina. The resulting samples were subjected to PCR amplification and incubated at 37 °C for 30 min. VAHTS DNA Clean Beads (Vazyme #N411) were used for purification and recovery of the DNA fragments. The purified product was amplified. VAHTS DNA Clean Beads were used for further purify and separate 200–700 bp fragments. The ATAC library was finally sequenced on a NextSeq 500, using a NextSeq 500 High Output Kit v2 (150 cycles) (FC-404-2002, Illumina), following the manufacturer's instructions.
3
Targeted Sequencing for Inherited Metabolic Diseases
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We performed targeted sequencing using the extended edition panel of inherited metabolic diseases (Genuine Diagnostic, Hanzhou, China) to detect 306 genes, including phenylalanine hydroxylase(PAH,MIM 612349), 6-pyruvoyltetrahydropterin synthase (PTS, MIM 612719), methylmalonyl-CoA mutase (MUT, MIM 609058), solute carrier family 22 member 5 (SLC22A5, MIM 603377) and so on (Supplementary Table 3 ). The target regions were enriched by multiple probe hybridization using an Agilent SureSelect Human Exon Sequence Capture Kit, and the capture products were purified using Agencourt AMPure XP Beads (Beckman Coulter, Brea, CA, USA). Purified products were treated with a TruePrep™ DNA Library Prep Kit V2 for Illumina (Vazyme Biotech Co., Ltd., Nanjing, China), and a special index was added using the TruePrep™ Index Kit V2 for Illumina (Vazyme). The quality of the DNA library was tested by Qubit and a 2100 Bioanalyser (Agilent High Sensitivity DNA Kit, Agilent Technologies, Santa Clara, CA, USA). Next, the sequencing libraries were quantified by using the Illumina DNA Standards and Primer Premix Kit (KAPA) and subjected to massively parallel sequencing on the Illumina HiSeq 2500 platform.
4
FFPE DNA Extraction and Exome Sequencing
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We extracted DNA from FFPE tissue samples using the Tissue Kit (69504, QIAGEN) following the manufacturer's protocols. Tissue sections were examined by two pathologists independently. Samples with at least 30% tumor cells were selected, 93% of which contained ≥50% tumor cells, and 86% contained ≥80% tumor cells. DNA was isolated by targeted capture pull‐down and exon‐wide libraries were generated from native DNA using the xGen® Exome Research Panel (Integrated DNA Technologies) and TruePrep DNA Library Prep Kit V2 for Illumina (#TD501, Vazyme). Paired‐end sequencing was performed by NovaSeq 6000. The sequencing data were then aligned to the human reference genome (NCBI build 37) using the Burrows–Wheeler Aligner (BWA), and polymerase chain reaction duplicates were sorted and removed by sambamba.
5
Single-cell RNA-seq Library Preparation
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Embryos were collected (5 embryos per sample) in 0.2 mL PCR tubes with a micro-capillary pipette and processed into cDNA with Superscript II reverse transcriptase as described described previously (Picelli et al., 2014 (link)). The cDNA is amplified with KAPA Hifi HotStart using 12 cycles. Sequencing libraries were constructed from 1 ng of pre-amplified cDNA using DNA library preparation kit (TruePrep DNA Library Prep Kit V2 for Illumina, Vazyme). Libraries were sequenced on a HiSeq-PE150, with paired end reads of 150 bp length each.
6
Whole Genome Sequencing of Bacterial Isolates
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Libraries were created by using TruePrep™ DNA Library Prep Kit V2 for Illumina®. WGS was carried out using the Illumina HiSeq X Ten PE150 with 150-base paired-end reads Quality of FASTQ-formatted sequencing reads was controlled with a minimum quality Phred score of 30 (as a rolling average over 4 bases with a minimum individual base quality of 15) using Trimmomatic-0.36 software (Bolger et al., 2014 (link)). In silico serotyping was performed using SISTR (Yoshida C. E. et al., 2016 (link)). SISTR classified the isolates into the corresponding serotype based on the serovar antigen combinations and core-genome multilocus sequence typing (cgMLST). The cgMLST type utilized by SISTR was obtained from EnteroBase. As SISTR requires contigs as the input file, the reads were assembled using shovill coupled with SPAdes v3.13.1 (Bankevich et al., 2012 (link)). Genome sequences obtained in this study have been submitted as raw reads under bioproject number PRJNA639393.
7
Single-cell RNA-seq of mouse embryos
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Embryos were collected from the mice of indicated genotypes (10 embryos per sample). Each sample was lysed with 4.2 μl lysis buffer (including 0.2 μl 1:1000 diluted ERCC spike-in) and was immediately used for cDNA synthesis using the Smart-seq2 method (Picelli et al., 2014). Briefly, cells were lysed in lysis buffer, and the polyadenylated mRNAs were captured usingby the PolyT primers. After 3 min lysis at 72°C, the Smart-seq2 reverse transcription reactions were performed. After the first-strand reaction, the cDNA wasis amplified using a limited number of cycles (∼13 cycles). Sequencing libraries were constructed from 500 pg of amplified cDNA using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD503) according to the manufacturer's instructions. Barcoded libraries were pooled and sequenced on the Illumina HiSeq X Ten platform in the 150 bp paired-end mode.
8
ATAC-seq of Mouse Erythroid Progenitor Cells
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ATAC-seq was performed as described previously (Wang et al., 2019 (link)), In brief, 50,000 MEP cells sorted from BM of flox/flox or Δ/Δ mouse cells were lysed in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% [vol/vol] IGPAL CA-630) for 10 min on ice. After centrifugation at 500 g for 5 min, the obtained nuclei were added to 50 μl transposition reaction buffer (offered by Vazyme TD501-01) followed with incubation at 37°C for 30 min. After tagmentation, VAHTS DNA Clean Beads were used to stop the reaction, and DNA was purified for final library construction (TruePrep DNA Library Prep Kit V2 for Illumina) before paired-end high-throughput sequencing using HiSeq XTe. Clean reads were aligned to the mouse genome (GRCm38) using BWA package, and peaks were called using MACS2 package (v2.2.5; Zhang et al., 2008 (link)) with a false discovery rate (FDR) cutoff of 0.05.
9
Micro-dissection and RNA-seq of epithelial cells
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Tissue samples from human or mice were cut into 5–10 consecutive sections (8 μm) and the epithelial layer contained 30–50 cells on each section was micro-dissected with a Leica LMD7000 laser-capture microdissection system. RNA was isolated from the mini-bulk samples and cDNA was prepared for sequencing based on the Geo-seq protocol59 (link). Sequencing libraries were built using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme), and evaluated by Bioanalyzer (DNA HS kit, Agilent). RNA-seq data were mapped to GRCh38 human genome and GRCm38 murine genome by HISAT2 (version 2.1.0)60 (link) with default parameter for human and murine samples, respectively. The gene expression matrix of raw reads counts after annotation by HTSeq (version 0.6.1p1) was processed using the DESeq2 (version 1.22.2)61 (link) and visualized by showing the first 3 dimensions calculated by plotPCA function. We used TRIZOL to extract bulk RNA from patient biopsy (n = 45), and constructed sequencing library using NEBNext Ultra II RNA Library Prep Kit for Illumina. Sequencing data were processed by HISAT2 and HTseq as described above. The normalized expression from bulk samples of human precancerous lesions was used to estimated neutrophil fraction with CIBERSORT (version 1.06)62 (link).
10
Transcriptomic Analysis of T Cell Activation
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RNA was isolated from activated splenic CD4+T cells from knockout (KO and WT mice. cDNA sequencing libraries were prepared using the TruePrep DNA Library Prep Kit V2 for Illumina and subjected to 2 × 150 paired-end sequencing. To identify differentially expressed genes (DEGs), fold expression changes were calculated for each gene by dividing the average fragments per kilobase of transcript per million mapped reads for the case by the average fragments per kilobase of transcript per million mapped reads for the control. The Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses were used to identify cellular pathways and biological processes associated with DEGs, respectively.
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