ChIP for ChIP-seq assays was performed as described above except that we used 2.5 μg antibodies and 1.5 × 107 ESCs for a transcription factor ChIP and 10 μg antibodies and 0.75 × 107 ESCs for a histone ChIP. The ChIPed and input DNA libraries were generated and sequencing was performed on an Illumina GAIIx or Hi-seq genome analyzer according to the manufacturer’s protocols. FAIRE-seq assays were essentially performed as described previously (Simon et al. 2012 (link)) with 2 × 106 ESCs per condition. Libraries for RNA-seq were generated with a SENSE mRNA-seq library kit (Lexogen) and sequencing was performed on an Illumina GAIIx genome analyzer according to the manufacturer’s protocols.
Hiseq genome analyzer
Manufactured by Illumina
Sourced in United States
The HiSeq Genome Analyzer is a lab equipment designed for high-throughput DNA sequencing. It utilizes advanced sequencing-by-synthesis technology to generate large volumes of high-quality sequencing data.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Gaiix, by Illumina (2 mentions)
Dna preparation kit and protocol, by Illumina (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Pronex purification system, by Promega (2 mentions)
Zymoclean gel dna recovery kit, by Illumina (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Turbo dna free kit, by Thermo Fisher Scientific (2 mentions)
Truseq technology, by Illumina (2 mentions)
Genome analyzer gaiix, by Illumina (1 mentions)
Sense mrna seq library kit, by Lexogen (1 mentions)
Ez dna methylation direct kit, by Zymo Research (1 mentions)
Zymoclean gel dna recovery kit, by Zymo Research (1 mentions)
Dna clean concentrator kit, by Zymo Research (1 mentions)
Dna preparation kit, by Illumina (1 mentions)
Dna clean concentrator, by Zymo Research (1 mentions)
Monarch rna cleanup kit, by New England Biolabs (1 mentions)
Ovation ultra low library prep kit, by Tecan (1 mentions)
Rneasy isolation kit, by Qiagen (1 mentions)
Casava 1, by Illumina (1 mentions)
17 protocols using hiseq genome analyzer
1
Chromatin and Transcriptome Profiling of ESCs
2
Genome-wide DNA Methylation Analysis
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To perform a genome-wide DNA methylation analysis, libraries were prepared from 200 to 500 ng of genomic DNA digested sequentially with 60 units of TaqI and 30 units of MspI (New England Biolabs, Ipswich, MA, USA). The resulting size-selected TaqI-MspI fragments (40–120 bp and 120–350 bp) were filled in, and 3′-terminal-A extended, extracted with DNA Clean & Concentrator™ kit (Zymo Research, Irvine, CA, USA). Ligation of selected fragments to pre-annealed adapters containing 5′-methyl-cytosine instead of cytosine was performed by using the Illumina DNA preparation kit in accordance with the protocol of the manufacturer (Illumina Inc., San Diego, CA, USA). Purified, adaptor-ligated fragments were then bisulphite-treated by using the EZ DNA Methylation-Direct™ Kit (Zymo Research). Preparative-scale PCR was performed with the resulting fragments followed by purification of PCR products with DNA Clean & Concentrator™ (Zymo Research). Final size selection of the purified PCR products was performed by using 4% NuSieve 3:1 agarose gel. SYBR-green-stained gel slices of adapter-ligated fragments (130–210 bp or 210–460 bp in size) were excised, and library material was recovered by using the Zymoclean™ Gel DNA Recovery Kit (Zymo Research). Sequencing was performed on an Illumina HiSeq genome analyzer.
3
EpiQuest DNA Library Preparation
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EpiQuest libraries were prepared according to Zymo Research Protocol (Irvin, CA). 200–500 ng of genomic DNA were digested with NEB 60 units of TaqI and 30 units of MspI (Ipswich, MA, USA) sequentially. Size-selected TaqI-MspI fragments (40–120 bp and 120–350 bp) were filled-in and 3′-terminal-A extended, extracted with Zymo Research (ZR) DNA Clean and Concentrator™-5 kit (Irvin, CA). Ligation to preannealed adapters containing 5′-methyl-cytosine was performed using Illumina's DNA preparation kit and protocol (San Diego, CA). Purified adaptor ligated fragments were bisulfite-treated using the EZDNA Methylation-Direct™ Kit (Irvin, CA). Preparative-scale PCR was performed. DNA Clean and Concentrator-purified PCR products were subjected to a final size selection on a 4% NuSieve 3 : 1 agarose gel. SYBR-green-stained gel slices containing adaptor-ligated fragments of 130–210 bp or 210–460 bp in size were excised. Library material was recovered from the gel (Zymoclean™ Gel DNA Recovery Kit, Irvin, CA, USA) and sequenced on an Illumina HiSeq Genome Analyzer (San Diego, CA).
4
RNA-seq Library Preparation and Sequencing
5
RNA-seq Transcriptome Profiling Protocol
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Total RNA was isolated employing the RNeasy Isolation Kit (Qiagen, Frederick, MD). Messenger RNA expression profiling was performed using NuGen reagents (www.nugeninc.com ). After amplification, libraries compatible with Illumina NGS methods were prepared using the Ovation Ultra Low Library Prep Kit (NuGen). The quality of each library was assessed using a Bioanalyzer 2100 (Agilent Technologies). Kappa Library Quant Kits (KappaBiosystems; www.kapabiosystems.com ) were used for library quantitation. Cluster generation and 2 × 36 bp paired-end sequencing was performed over a single lane using the Illumina GAIIx or HiSeq Genome Analyzer workstation. The RNA-Seq data were analyzed and quantified employing Cufflinks [18] (link).
6
Exome Sequencing of Tumor and Normal DNA
7
Illumina Library Preparation and Sequencing
8
Tagmentation and Library Preparation from DNA
9
ATAC-seq for Chromatin Profiling
10
Transcriptome Analysis of Cortical Development
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RNAseq analysis was carried out on polyA + RNA from two brains each of Nes-KO and wild-type littermates at E18, and dissected mouse cortices from Emx-KO and wild-type littermates at P1. Paired end libraries for 100 nt read length were constructed using the Illumina Tru-seq kit. The Emx-KO libraries were strand specific with the second cDNA strand eliminated with the USER enzyme (Bhatt et al., 2012 (link)). Libraries were sequenced on an Illumina HiSeq Genome analyzer in the sequencing core at the Broad Stem Cell Research Center at UCLA. Data were analyzed using the standard TopHat, Cufflinks, Cuffmerge, and Cuffdiff pipeline generating 312M and 327M mapped reads from NesKO and wild-type mice respectively at E18 (Trapnell et al., 2012 (link)). At P1, EmxKO and wild-type samples generated 354M and 317M mapped reads respectively. Alternative exon inclusion levels were determined by additional mapping to an exon duo and trio database using Splicetrap (Wu et al., 2011 (link)). Gene Ontology Analysis was performed using DAVID (Huang da et al., 2009 (link)).
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