dRNA-Seq libraries were prepared as previously described [28 (link)]. About 700 ng rRNA-depleted RNA was incubated in 1× RNA 5′ polyphosphatase (TAP) (Epicentre) reaction buffer and 1 U of SUPERase-In (Invitrogen) at 37 °C for 1 h with [TAP(+)] or without [TAP(−)] 1 U of TAP. After ethanol precipitation, 5 pmol of 5′ RNA adaptor (5′-ACACUCUUUCCCUACACGACGCUCUUCCGAUCU-3′) was ligated to the purified RNA with T4 RNA ligase (Thermo) in 1× RNA ligase buffer and 0.1 mg/mL BSA by incubating at 37 °C for 90 min. The adaptor-ligated RNA was then purified using Agencourt AMPure XP beads (Beckman Coulter) according to the manufacturer’s instructions. The purified product was reverse-transcribed using SuperScript III Reverse Transcriptase (Invitrogen) and purified using Agencourt AMPure XP beads. The purified cDNA was amplified and indexed using Phusion High-Fidelity DNA Polymerase (Thermo) for the Illumina sequencing. The amplification step was monitored using a CFX96 Real-Time PCR Detection System (Bio-Rad) and stopped before the PCR reaction was fully saturated. Finally, the amplified library was purified using Agencourt AMPure XP beads, and the concentration of the library was measured with Qubit 2.0 fluorometer (Invitrogen). The size distribution of the library was checked with gel electrophoresis on 2% agarose gel.
Agencourt ampure xp beads
Manufactured by Thermo Fisher Scientific
Sourced in United States
Agencourt Ampure XP beads are magnetic beads used for the purification of DNA and RNA samples in various molecular biology applications. They provide a simple and efficient way to separate target biomolecules from contaminants, reaction components, and unwanted fragments. The beads can be used with a wide range of sample types and are compatible with various automated liquid handling systems.
Automatically generated - may contain errors
Lab products found in correlation
Agencourt ampure xp bead, by Beckman Coulter (4 mentions)
Nd 1000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Phusion high fidelity dna polymerase, by Thermo Fisher Scientific (1 mentions)
T4 rna ligase, by Thermo Fisher Scientific (1 mentions)
Superase in, by Thermo Fisher Scientific (1 mentions)
Superscript 3 reverse transcriptase, by Thermo Fisher Scientific (1 mentions)
Qubit 2.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Cfx96 real time pcr detection system, by Bio-Rad (1 mentions)
Dynabeads myone streptavidin t1, by Thermo Fisher Scientific (1 mentions)
Novaseq, by Illumina (1 mentions)
E gel, by Thermo Fisher Scientific (1 mentions)
Pe1.0 and pe2 0 primers, by Illumina (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Phusion high fidelity pcr master mix, by New England Biolabs (1 mentions)
S220 focused ultrasonicator, by Covaris (1 mentions)
Dynabeads myone streptavidin t1 beads, by Thermo Fisher Scientific (1 mentions)
Sureselectxt reagent kit, by Illumina (1 mentions)
Bioanalyzer dna 1000 chip, by Beckman Coulter (1 mentions)
Hiseq paired end sequencing library, by Illumina (1 mentions)
11 protocols using agencourt ampure xp beads
1
Preparation of dRNA-Seq Libraries
2
DNA Fragmentation and Size Selection
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2 µg of DNA was sheared with a Covaris E220 focused-ultrasonicator with the following settings: 10% duty cycle, intensity 5, 200 cycles per burst, 150 s. DNA was split into two aliquots (1 µg each) and samples were size-selected for fragments of 300 bp using 0.85X Agencourt AMPure XP beads (Invitrogen) with a 1.4x ratio following the manufacturer’s instructions.
3
Exome Sequencing Protocol for Clinical Genetics
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The next-generation sequencing was performed by the laboratory of GenePhile Bioscience (Taiwan). To generate standard exome capture libraries, SureSelect XT Human All Exon Clinical Exome version 2 (CREv2, 66Mb) probe set was used (Agilent Technology, USA). For DNA library construction, 1 μg genomic DNA was used with Agilent SureSelect XT Reagent kit. The amplification adapter-ligated sample was purified by utilizing Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and then analyzed on a TapeStation 4200 D1000 screentape. Seven hundred fifty nanograms of the gDNA library was prepared for hybridization with the capture baits. The sample was hybridized at 65°C for 24 h, captured with the Dynabeads MyOne Streptavidin T1 (Life Technologies, USA) and purified using Agencourt AMPure XP beads. Use the Agilent protocol to addition of index tags by posthybridization amplification. The sample was sequenced on Illumina NovaSeq with 150PE protocol. The qualified reads data then went through a genomic alignment against Ensembl database using Burrows-Wheeler Aligner (BWA 0.7.15) to get basic sequence information. To further the variants analysis, variants calling and annotations were created by Genome Analysis Toolkit (GATK 3.0) and Variant Effect Predictor (VEP86).
4
Mitochondrial DNA Library Preparation
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The mitochondrial DNA libraries were generated using a protocol adapted from Wilkening et al. (2013) (link). One to 10 µg of mitochondrial DNA was sheared using the S220 Focused ultrasonicator (Covaris) with the following conditions: duty cycle, 20%; intensity, 5; cycles per burst, 200; duration, 45 sec; temperature, 4°. The 350-bp fragments were purified using 1 vol Agencourt AMPure XP beads (Beckman Coulter). The fragments were submitted to end repair, A addition, and adapter ligation with enzyme heat inactivation after each step. The samples were purified using 1 vol Agencourt AMPure XP beads and the libraries were amplified by PCR (98° for 45 sec; 12 cycles of 98° for 15 sec, 65° for 30 sec, and 72° for 30 sec; and 72° for 5 min). This step was performed using 2–10 ng of DNA, 2× Phusion High Fidelity PCR Master Mix (New England Biolabs, Beverly, MA), and 0.2 µM PE1.0 and PE2.0 primers (Illumina, San Diego, CA). The PCR products were then cleaned with 1 vol Agencourt AMPure XP beads and the yield was quantified with a Qubit Fluorometer (Life Technologies, Carlsbad, CA). Up to 13 samples were pooled and purified for 400- to 450-bp fragments on an E-gel (Life Technologies, Carlsbad, CA). The multiplexed libraries were sequenced on a Hiseq 2000, yielding between 2.8 and 42 million reads per sample (Table S2 ).
5
Standardized Exome Capture Library Generation
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For the generation of standard exome capture libraries, we used the Agilent SureSelect XT Reagent Kit protocol for an Illumina Hiseq paired-end sequencing library (catalog#G9611A). In all cases, the SureSelect XT Human All Exon Version 6 (60 Mb) probe set was used. We used 1000 ng genomic DNA to construct each library. Each adapter-ligated sample was purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA) and analyzed on a Bioanalyzer DNA1000 chip. A total of 750 ng of the sample was prepared for hybridization with the capture baits, and the sample was hybridized for 90 min at 65 °C, captured with Dynabeads MyOne Streptavidin T1 beads (Life Technologies, USA), and purified using Agencourt AMPure XP beads. We used the Agilent protocol to add index tags by post-hybridization amplification. Finally, all samples were sequenced on an Illumina Hiseq4000 instrument using the 150PE protocol.
6
Microsatellite Amplicon Sequencing Protocol
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PCR (20 ng of DNA) and LT-RPA (5 ng of DNA, 10.5 mM of Mg(OAc)2 and 40 min incubation at 32°C) amplicons of HT17, NR24, CAT25, BAT26, D2S123, D18S61, D12ATA63, REN and HPRTII microsatellites (shorter primers were used for REN and HPRTII and are indicated in Supplementary Table S1 ) obtained for each blood sample were purified using Beckman Coulter™ Agencourt AMPure XP beads (Thermo Fischer Scientific), quantified and pooled in equimolar ratio. 900 ng of each pool were used for the ligation of dual-indexed adapters using QIAseq 1-Step Amplicon Library Kit (Qiagen) and no supplemental PCR amplification of the libraries was required. Libraries were assessed for quality and quantity with a Fragment Analyzer (Agilent) and QIAseq™ Library Quant Assay Kit (Qiagen) respectively. 50 pM of the pooled libraries were deposited on an iSeq 100 cartridge (Illumina) together with 20% of 50 pM PhiX control v3 Library (Illumina). Amplicon sequencing was performed on an iSeq 100 using 151 cycles of paired-end sequencing. FastQ files were generated for each sample using Local Run Manager Software (Illumina) and the read counts and sequences of each microsatellite allele were obtained from the aligned reads using an in-house developed Python code.
7
Ribo-depletion and RNA-seq Library Prep
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Total RNA was ribo-depleted with Low Input RiboMinus Eukaryote System v2 (Thermo Fisher Scientific, A15027). RiboMinus Magnetic Bead Cleanup (Thermo Fisher Scientific, A15026) was used to concentrate and purify the rRNA-depleted RNA. Ion Total RNA-Seq Kit v2 was used to make cDNA, add barcodes, and amplify the library. All cleanup steps were performed using Agencourt Ampure XP beads (Thermo Fisher Scientific, A63880).
8
Ribosomal RNA Depletion and Sequencing of Archaeal Transcriptome
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A Ribo-Zero rRNA Removal Bacteria kit (Epicenter, Madison, WI) used with manufacturer’s protocol was applied to deplete a maximum of rRNAs and enrich total RNA in mRNA. As it is often the case with Archaea, ribodepletion attempts were poorly efficient and the RNA-seq dataset still contained 73–94% ribosomal sequences. Ion Total RNA-Seq Kit v2 was used to make cDNA, to add barcodes, and to amplify the library. All cleanup steps were performed using Agencourt Ampure XP beads (Thermo Fisher Scientific). Each step was validated by a bioanalyzer quality control on Agilent RNA chips and/or with the “QubitTM” fluorometer (Life Technologies) to determine the quality, size of fragments and quantity of produced material. RNA libraries were sequenced on P1v2 chips using the Ion S5TM System (Thermo Fisher Scientific). Sequencing was completed by the GeT-Genotoul platform in Toulouse (France). The RNA-seq data have been deposited into the NIH Sequence Read Archive (SRA) under the BioProject ID PRJNA739722 (accession numbers SRX11191114 -11191122 ).
9
Whole Exome Sequencing using Ion Proton
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We performed WES using an Ion Proton System equipped with a PI chip V2 together with an AmpliSeq Exome kit (Thermo Fisher Scientific) [26 (link)]. Briefly, 100 ng each of tumor and matched blood cell DNA was used for target amplification with the following protocol: 99 °C for 2 min, followed by 10 cycles at 95 °C for 15 s and 60 °C for 16 min, and a final hold at 10 °C. The incorporated primer sequences were partially digested using FuPa Reagent (Thermo Fisher Scientific). Ion Torrent Proton adapters were ligated to the amplicons at 22 °C for 30 min and then at 72 °C for 10 min. The amplicon library was purified using Agencourt AMPure XP Beads (Thermo Fisher Scientific). The library DNA was quantified by qRT-PCR, and 7 pM library DNA was used for sequencing. The sequencing data were aligned to the human reference genome (assembly GRCh37/hg19) and were quality trimmed using Ion Torrent Suite version 4.2 (Thermo Fisher Scientific). The mutations were visualized using the Integrative Genomics Viewer [27 (link)] and were validated using Sanger sequencing or pyrosequencing.
10
Automated RNA-seq Library Preparation
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RNA extraction was performed using the Qiagen RNeasy kit (Qiagen, Manchester, UK) according to the manufacturer’s instructions. The samples were checked for RNA quality using the Agilent 2100 Bioanalyzer (Agilent Technologies UK Limited, Craven Arms, UK) and quantified using a Nanodrop instrument (ND-1000 Spectrophotometer; Thermo Fisher Scientific Inc.). The extracted RNA was subsequently subjected to treatment with DNAse I (Thermo Fisher Scientific Cat. no: AM222) followed by purification with Agencourt Ampure XP beads (Thermo Fisher Scientific Cat. no: A63881). A 20 ng aliquot of intact high-quality total RNA (RIN > 9) from each sample was then used as input to generate libraries for RNA-seq using the NEBNext Ultra II Directional RNA kit (NEB, Hitchin Herts, UK; Cat. no: E7760S) following the manufacturer’s recommendations. This protocol involved an initial step of rRNA depletion, followed by fragmentation prior to first-strand cDNA synthesis and barcoding of the second-strand cDNA synthesised with indices for Illumina platform sequencing for final library amplification. The resulting libraries were assessed on the Bioanalyzer 2100 for purity. Sequencing of the resultant double-stranded (ds) cDNA libraries was performed by applying Illumina sequencing by synthesis technology.
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