Total RNA was isolated from 5 million B-LCLs using RNeasy mini kit including DNase I treatment (Qiagen) according to the manufacturer’s instructions. Total RNA was quantified using the NanoDrop 2000 (Thermo Scientific) and RNA quality was assessed with the 2100 Bioanalyzer (Agilent Technologies). Transcriptome libraries were generated from 1 μg of total RNA using the TruSeq RNA Sample Preparation Kit v2 (Illumina) following the manufacturer’s protocol. In brief, poly-A messenger RNA was purified using poly-T oligo-attached magnetic beads using two rounds of purification. During the second elution of the poly-A RNA, the RNA was fragmented and primed for cDNA synthesis. Reverse transcription of the first strand was performed using random primers and SuperScript II (Invitrogen). A second round of reverse transcription was done to generate a double-stranded cDNA, which was then purified using Agencourt AMpure XP PCR purification system (Beckman Coulter). End repair of fragmented cDNA, adenylation of the 3′ ends and ligation of adaptors were completed following the manufacturer’s protocol. Enrichment of DNA fragments containing adapter molecules on both ends was done using 15 cycles of PCR amplification and the Illumina PCR mix and primers cocktail.
Agencourt ampure xp pcr purification system
Manufactured by Beckman Coulter
Sourced in United States
The Agencourt AMPure XP PCR purification system is a magnetic bead-based technology for the purification of PCR amplicons. It allows for the efficient removal of primers, nucleotides, salts, and other contaminants from PCR reactions.
Automatically generated - may contain errors
Lab products found in correlation
Miseq platform, by Illumina (2 mentions)
Nextseq 500, by Illumina (2 mentions)
Dna clean concentrator 5 kit, by Zymo Research (2 mentions)
Nextera dna sample prep kit, by Illumina (2 mentions)
Nextseq 500 high output kit, by Illumina (2 mentions)
Epitect bisulfite kit, by Qiagen (2 mentions)
Takara ex taq dna polymerase, by Takara Bio (2 mentions)
Takara extaq pcr buffer 10x, by Takara Bio (2 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Superscript 2, by Thermo Fisher Scientific (1 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Dnase 1 treatment, by Qiagen (1 mentions)
Truseq rna sample preparation kit v2, by Illumina (1 mentions)
Q5 high fidelity polymerase, by New England Biolabs (1 mentions)
Nextflex 16s v4 amplicon seq kit, by PSC Biotech (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Quant it picogreen dsdna reagent, by Thermo Fisher Scientific (1 mentions)
454 gs junior titanium platform, by Roche (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
16 protocols using agencourt ampure xp pcr purification system
1
RNA-seq Transcriptome Library Preparation
2
16S rRNA Gene Amplification and Sequencing
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DNA from 48 tissue sample pairs (tumor and healthy mucosa) and 129 fecal samples (which are a
subset of study population F) was amplified using primers targeting the V4 region of the 16S rRNA
gene (F515 5′-GTGCCAGCMGCCGCGGTAA-3′, R806 5′-GGACTACHVGGGTWTCTAAT-3′)
(Caporaso et al, 2011 (link)). PCR was carried out
according to the manufacturer's instructions of the Q5 high-fidelity polymerase (New England
BioLabs, Ipswich, USA) using bar-coded primers (NEXTflex™ 16S V4 Amplicon-Seq Kit, Bioo
Scientific, Austin, Texas, USA) at final concentrations of 0.2 μM and an annealing
temperature of 56°C for 35 cycles.
PCR products were cleaned up with Agencourt AMPure XP-PCR Purification system (Beckman Coulter,
Brea, USA), quantified according to the NEXTflex™ 16S V4 Amplicon-Seq Kit protocol, and
multiplexed at equal concentration. Sequencing was performed using a 250-bp paired-end sequencing
protocol on the Illumina MiSeq platform (Illumina, San Diego, USA) at the Genomics Core Facility,
European Molecular Biology Laboratory, Heidelberg.
subset of study population F) was amplified using primers targeting the V4 region of the 16S rRNA
gene (F515 5′-GTGCCAGCMGCCGCGGTAA-3′, R806 5′-GGACTACHVGGGTWTCTAAT-3′)
(Caporaso et al, 2011 (link)). PCR was carried out
according to the manufacturer's instructions of the Q5 high-fidelity polymerase (New England
BioLabs, Ipswich, USA) using bar-coded primers (NEXTflex™ 16S V4 Amplicon-Seq Kit, Bioo
Scientific, Austin, Texas, USA) at final concentrations of 0.2 μM and an annealing
temperature of 56°C for 35 cycles.
PCR products were cleaned up with Agencourt AMPure XP-PCR Purification system (Beckman Coulter,
Brea, USA), quantified according to the NEXTflex™ 16S V4 Amplicon-Seq Kit protocol, and
multiplexed at equal concentration. Sequencing was performed using a 250-bp paired-end sequencing
protocol on the Illumina MiSeq platform (Illumina, San Diego, USA) at the Genomics Core Facility,
European Molecular Biology Laboratory, Heidelberg.
3
ATAC-seq Library Preparation from Murine Nuclei
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All ATAC-seq experiments were performed with 3 biologically independent mice per experimental condition. ATAC-seq was performed as previously described47 . 50,000 unfixed nuclei were tagged using Tn5 transposase (Nextera DNA sample prep kit; Illumina) for 30 min at 37°C. Libraries were generated using the Ad1_noMX and Ad2.1-24 barcoded primers48 (link) and amplified for 10-12 cycles. Resulting library fragments were purified using a DNA Clean & Concentrator-5 kit (Zymo Research). Size-selection was performed using the Agencourt AMPure XP PCR purification system (Beckman Coulter) to remove large fragments > 1 kb and primers to maintain high library complexity. Libraries qualities were assessed using the Agilent Bioanalyzer High-Sensitivity DNA kit and sequenced on an Illumina NextSeq500 with a read length of 75 bp to a minimum depth of 40 million reads per sample using the NextSeq 500 High-Output Kit (150-Cycle, 400 M Flow Cell).
4
Amplicon Sequencing on 454 GS Junior
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PCR amplicons were purified from gel using the QIAquick gel extraction kit (Qiagen) and extracted DNA was again purified using Agencourt AMPure XP PCR purification system (Beckman Coulter). The quality and length of the amplicons were verified using Agilent 2100 bioanalyzer (Agilent Life Science, Santa Clara, California) and the concentration was quantified using Quant-iT PicoGreen dsDNA Reagent (Invitrogen) on a TECAN fluorometer (TECAN infinite F200). After quantification, amplicons were pooled in equimolar concentrations, followed by emulsion PCR (emPCR) and bead enrichment according to the 454 Titanium emPCR and enrichment bead recovery protocols (Roche), according to instructions of the manufacturer. The enriched beads were sequenced in both forward and reverse direction on the 454 Life Science platform (454 GS Junior, Roche Applied Science) according to the manufacturer’s instructions.
5
Colonic Microbiome Profiling via 16S rRNA Sequencing
6
Targeted Enrichment of DNA Libraries
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The DNA libraries are prepared using the Thru PLEX Tag-Seq HV kit (Takara Bio Inc. Japan) as per the manufacturer’s instructions. The hybrid capture is performed using IDT xGen Hybridization and Wash Kit (IDT Inc. USA) protocol. Streptavidin-coated magnetic beads are used to bind and extract biotinylated probe-hybridized target DNA. This protocol includes target enrichment by PCR prior to sequencing using KAPA HiFi HotStart Ready Mix and DNA clean up using Agencourt AMPure XP PCR Purification system (Beckman Coulter, USA). The capture library products are assessed using Qubit 3.0 Fluorometer (Thermo Fisher Scientific, USA) and Agilent Bioanalyzer High Sensitivity DNA assay (Agilent Technologies Inc., USA).
7
Bisulfite Conversion and Nested PCR for DNA Methylation Analysis
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A total of 1.5 µg of brain DNA [23 (link)] was bisulphite-converted using the QIAGEN Epitect® Bisulfite Kit, as per the manufacturer’s protocol (Hilden, Germany). With this kit we typically perform two consecutive treatments to avoid incomplete conversion, especially in GC-rich regions [35 (link)]. The converted DNA was amplified via a nested PCR reaction (40 cycles) with gene specific primers (the bisulfite-sequencing primers have been described elsewhere [21 (link)]). The PCR products were purified utilising an Agencourt® AMPure® XP PCR Purification system (Beckman Coulter, Brea, CA, USA).
8
16S rRNA Gene Sequencing Protocol
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In order to obtain the 16S rRNA gene sequencing of the participant’s DNA samples, we performed a PCR-amplified using the hypervariable V4 region of this gene with universal bacteria/archaeal primers 515F/806R following the procedures reported by Caporaso et al.62 (link), Carrillo et al.63 (link) and Osiris-Gaonaet al.64 . PCR reactions (25 l) contained 2–6 ng of total DNA, 2.5 l Takara ExTaq PCR buffer 10X, 2 l Takara dNTP mix (2.5 mM), 0.7 l bovine serum albumin (BSA, 20 mg ml ), 1 l primers (10 M), 0.125 l Takara Ex Taq DNA Polymerase (5 U l-1) (TaKaRa, Shiga, Japan) and nuclease-free water. All samples were amplified in triplicate implementing a PCR protocol that includes an initial denaturation step at 95 C (3 min), 35 cycles of 95 C (30 s), 52 C (40 s), 72 C (90 s), and a final extension (72 C, 12 min). The triplicates were pooled and purified using the SPRI magnetic bead, AgencourtAMPure XP PCR purification system (Beckman Coulter, Brea, CA, USA), and the 16S rRNA fragments ( 20 ng per sample) were sequenced on an IlluminaMiSeq platform (Yale Center for Genome Analysis, CT, USA), generating 250 bp paired-end reads. All sequence data are available from the NCBI Bioproject number PRJNA593240.
9
Bisulfite Conversion and Nested PCR for amegfr Analysis
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1.5 μg of larval genomic DNA28 (link) was bisulfite converted using the QIAGEN Epitect® Bisulfite Kit, as per the manufacturer’s protocol29 . We routinely perform two consecutive treatments to avoid incomplete conversion especially in GC-rich regions. The converted DNA was amplified via a nested PCR reaction with amegfr specific primers (see Table S2 for BS-seq primers). The PCR products were purified utilising Agencourt® AMPure® XP PCR Purification system (Beckman Coulter).
10
Illumina RNA-Seq Library Prep Protocol
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For sequencing on Illumina platforms, cDNA libraries were synthesized using the SMARTer Stranded Total RNA-Seq Kit – Pico Input Mammalian (TaKaRa Bio Inc., Kusatsu, prefecture Shiga, Japan). Briefly, 10 ng of total RNA was fragmented according to manufacturer’s instructions and used for first-strand synthesis followed by addition of Illumina adapters. Ribosomal cDNA was depleted and the final double-stranded cDNA library was amplified via PCR according to manufacturer’s instructions. Before depletion of ribosomal cDNA and after PCR amplification, the double-stranded cDNA libraries were purified using the Agencourt AMPure XP PCR purification system (Beckman Coulter, Brea, CA, USA). The first purification step was performed according to the manufacturer´s instructions. The second purification step contained minor modifications. Briefly, the amount of beads was increased to 100 μl volume and the cDNA was eluted in 20 μl elution buffer. To quantify the double-stranded cDNA libraries, the Qubit dsDNA HS Assay Kit and the Qubit Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) were used.
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