16S library preparation for DNAs from all groups was performed per our published protocol.23 (link) An optimised clean-up step was performed with Agencourt AMPure XP beads using modifications proposed by Quail et al,24 (link) and 5 µL of the final Amplicon PCR product was used for the Index PCR, which was performed using the Nextera XT Index kit and 2 x KAPA HiFi HotStart Ready Mix, following the Illumina protocol. After a second modified clean-up step, the final product was quantified using Qubit ds DNA BR Assay kits and Qubit 1X dsDNA HS Assay kits on a Qubit 4 Fluorometer, and analysed using Agilent DNA 1000 chips (Agilent Technologies, Santa Clara, California, USA) and Agilent HS DNA chips (Agilent) on an Agilent 2100 Bioanalyzer System.
Agilent hs dna chip
Manufactured by Agilent Technologies
Sourced in United States
The Agilent HS DNA chip is a high-sensitivity DNA microarray for gene expression analysis. It provides reliable and accurate measurement of gene expression levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500, by Illumina (2 mentions)
Nextera xt dna sample preparation kit, by Illumina (2 mentions)
Hiseq instrument, by Illumina (2 mentions)
Megaclear column, by Thermo Fisher Scientific (2 mentions)
Dnase treatment, by Thermo Fisher Scientific (2 mentions)
Microbexpress, by Thermo Fisher Scientific (2 mentions)
Absolute qpcr sybr green rox mix, by Thermo Fisher Scientific (2 mentions)
Rnaseh and e coli dna polymerase, by New England Biolabs (2 mentions)
Superscript 2 and random hexamers, by Thermo Fisher Scientific (2 mentions)
Kapa hifi hotstart readymix, by Illumina (1 mentions)
Nextera xt index kit, by Illumina (1 mentions)
Agilent dna 1000 chip, by Agilent Technologies (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Qubit 1x dsdna hs assay kits, by Agilent Technologies (1 mentions)
Qubit 4 fluorometer, by Agilent Technologies (1 mentions)
Novaseq platform, by Illumina (1 mentions)
Countess 2, by Thermo Fisher Scientific (1 mentions)
Dynabeads myone silane beads, by Thermo Fisher Scientific (1 mentions)
Single cell a chip kit, by 10x Genomics (1 mentions)
6 protocols using agilent hs dna chip
1
16S DNA Library Preparation with Optimized Cleanup
2
Single-Cell RNA-Seq of Cell Suspensions
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Cell suspensions were submitted to the University of Wisconsin (UW) Biotechnology Center (UWBC) for processing. Collected cells were concentrated and cell viability was further validated using the Countess ™ II (Invitrogen). A total of 56,000 cells (7000 for each of 8 samples) were targeted using the 10X Genomics V(D)J Single-Cell RNA-Seq pipeline. Briefly, GEMs were prepared using the Single Cell A Chip Kit (10X Genomics). Full-length cDNA cleanup was performed using DynaBeads Myone Silane beads (Invitrogen). Full-length cDNA was then amplified by PCR (13 cycles) and post cDNA amplification cleanup was performed using SPRIselect (Beckman Coulter) and QC was performed using Agilent HS DNA chips. Feature barcode (FB) PCR amplification was done in 9 cycles. Sample indexing was then performed using the Chromium i7 Plate N, Set A indices (10X Genomics). Gene expression (GEX), FB, and V(D)J libraries were then prepared respectively. Libraries meeting all quality control criteria were then sequenced using the Illumina NovaSeq platform at the UW Gene Expression Center in collaboration with the UWBC DNA Sequencing Facility, Madison, Wisconsin. Libraries were sequenced at the following depth (reads/cell): GEX: 85,000; V(D)J: 11,000; FB: 6,400, with a total of 53,000 cells submitted for sequencing.
3
Illumina RNA-Seq Library Preparation Protocol
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Total RNA was amplified using the Ovation RNA-Seq System v2 assay (Tecan Genomics) according to the manufacturer’s protocol. The libraries were prepared with the Nextera XT DNA Sample Preparation kit (Illumina Technologies). In total, 1 ng of cDNA was resuspended in Tagment DNA Buffer, and tagmentation (fragmentation and tagging with the adaptors) was performed with the Nextera enzyme (Amplicon Tagment Mix), incubating at 55°C for 10 minutes. Neutralize Tagment (NT) buffer was then added to neutralize the samples (part of the Nextera XT DNA Sample Preparation kit, Illumina Technologies). Libraries were prepared by PCR with the random primer Nextera PCR Master Mix and 2 Nextera Indexes (N7XX and N5XX) according to the following program: 1 cycle of 72°C for 3 minutes; 1 cycle of 98°C for 30 seconds; 12 cycles of 95°C for 10 seconds, 55°C for 30 seconds, and 72°C for 1 minute; and 1 cycle of 72°C for 5 minutes. The size of the libraries for each sample was measured using the Agilent HS DNA chip (Agilent Technologies). The samples were placed in a pool. The concentration of the pool was optimized to acquire at least 25 million to 30 million reads per sample. Libraries were sequenced on the Illumina HiSeq2500 following the manufacturer protocol using Paired-End Reads with a read length of 75 bps. RNA-Seq analysis was performed by BioWardrobe, as previously described (56 (link)).
4
Microbial Community DNA and mRNA Sequencing
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Methods for microbial community DNA and mRNA sequencing were as previously described 44 (link),45 (link). Fecal pellets collected from mice before or after LPS treatment (pooled from 2 and 3 days post-injection) were kept at −80°C until DNA and RNA isolation using the guanidinium thiocyanate/cesium chloride gradient method 46 (link) as described, except that crude particles were removed by centrifugation prior to overlaying the gradient. RNA was subjected to DNase treatment (Ambion), purification using MEGAClear columns (Ambion), and rRNA depletion via subtractive hybridization (MICROBExpress, Ambion, in addition to custom depletion oligos). The presence of genomic DNA contamination was assessed by PCR with universal 16S rDNA primers. cDNA was synthesized using SuperScript II and random hexamers (Invitrogen, Carlsbad, CA), followed by second strand synthesis with RNaseH and E.coli DNA polymerase (New England Biolabs). Samples were prepared for sequencing with an Illumina HiSeq instrument after enzymatic fragmentation (NEBE6040L/M0348S). Libraries were quantified by quantitative PCR (qPCR) according to the Illumina protocol. qPCR assays were run using ABsoluteTM QPCR SYBR Green ROX Mix (Thermo Scientific) on an Mx3000P QPCR System instrument (Stratagene, La Jolla, CA). The size distribution of each library was quantified on an Agilent HS-DNA chip (Agilent, Santa Clara, CA).
5
Isolating Alveolar Macrophages from Mouse Lungs
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To isolate in-vivo-derived alveolar Mφ, C57BL/6 CD45.1 mice were sacrificed and bronchoalveolar lavage was performed to isolate SIGLEC-F+ cells via fluorescence-activated cell sorting (FACS). PMT-Mφ were recovered from transplanted animals based on CD45.1 expression by FACS from BALF and lungs. In addition, iPSCs, BM-, and iPSC-Mφ from in vitro differentiation were harvested by trypsinization. For all cells, RNA was isolated using the RNAeasy Plus Micro Kit (QIAGEN). Total RNA (10 ng) was amplified using the Ovation RNA-Seq System v.2 (NuGEN) according to the manufacturer's protocol. The libraries were prepared with the Nextera XT DNA Sample Preparation Kit (Illumina) and the concentrations were measured using the Qubit dsDNA HS (High Sensitivity) assay. The size of the libraries for each sample was measured using the Agilent HS DNA chip. The libraries were sequenced on an Illumina HiSeq 2500 in Rapid Mode, generating 20 million or more high-quality 75-base-paired long end reads per sample in the Gene Expression Core Facility and DNA Sequencing Core in Cincinnati Children's Hospital Medical Center.
6
Microbial Community DNA and mRNA Sequencing
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