To the cDNA, 3 µL 10X CircLigase Buffer (Epicentre), 1.5 µL 50 mM MnCl2, 1.5 µL 1 mM ATP, 0.5 µL 100 µM DNA adapter, oligonucleotide C (Supplementary Table 4 ), and 1 µL CircLigase I (Epicentre) were added. The reaction was incubated at 60 °C for 2 hr, then 80 °C for 10 min to inactivate the ligase. The ligated DNA was EtOH precipitated with 1 µL 20 mg/mL glycogen as a carrier and dissolved in 20 µL of nuclease-free H2O. Then the cDNA was purified using 36 µL of Agencourt XP beads (Beckman Coulter), according to manufacturer’s instructions and eluted with 20 µL TE buffer.
Agencourt xp beads
Manufactured by Beckman Coulter
Sourced in United States, Germany
Agencourt XP beads are magnetic microparticles used for nucleic acid purification in molecular biology applications. They provide a flexible and efficient solution for the isolation and purification of DNA and RNA from a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Circligase 1, by Illumina (5 mentions)
Circligase buffer, by Illumina (5 mentions)
Mastercycler pro, by Eppendorf (2 mentions)
Ercc exfold rna spike in mix 2, by Thermo Fisher Scientific (2 mentions)
Fragment library construction kit, by Thermo Fisher Scientific (2 mentions)
Solid 4.0 sequencer, by Thermo Fisher Scientific (2 mentions)
Qubit system, by Thermo Fisher Scientific (1 mentions)
Miseq system, by Illumina (1 mentions)
E gel, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
2100 bioanalyzer high sensitivity dna kit, by Agilent Technologies (1 mentions)
Kapa hifi hotstart mastermix, by Avantor (1 mentions)
Nextera xt dna library preparation, by Illumina (1 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (1 mentions)
Kapa hifi hotstart readymix, by Roche (1 mentions)
Sureselectxt mouse all exon kit, by Agilent Technologies (1 mentions)
Agilent bioanalyzer high sensitivity dna kit, by Agilent Technologies (1 mentions)
Qubit assay, by Thermo Fisher Scientific (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Herculase 2 fusion dna polymerase, by Agilent Technologies (1 mentions)
18 protocols using agencourt xp beads
1
cDNA Circularization and Purification
2
Construction of LeGO-G2-BC16/BC32 Plasmid Library
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We constructed a LeGO-G2-BC16 plasmid library, containing an eGFP as a fluorescent reporter and the GFP-BC16 as barcode sequence, according to the recently described RGB-barcode vectors5 (link). For the GFP-BC32 construct, the original LeGO-vector backbone (LeGO-G2)24 (link) was additionally equipped with a truncated sequence for the Illumina-Indexed-Adaptor and unique restriction enzyme recognition sites for XbaI and XhoI (LeGO-G2-BC32). The GFP-BC32 forward oligonucleotide (Poly-GFP-Barcode-Fw) consists of 32 wobble bases interspersed by fixed triplets in a defined order and of a modified version of the Illumina-Truseq Universal-Adapter-Sequence. Additionally, we included three wobble bases upstream of the barcode to prevent phasing of the Illumina system. The barcode was manufactured by TIB Molbiol. Second-strand synthesis was performed by using primer 32-BC-Poly-Rv with 500 ng of Poly-GFP-Barcode-FW. Obtained double strands were purified using Agencourt XP-beads (Beckman Coulter) and digested with XbaI and XhoI. To exclude fragmentary barcodes, a purification step via gelextraction was performed prior to ligation into the XbaI/XhoI-digested LeGO-G2-BC32 backbone. Ligation of the BC32-oligonucleotides and the vector backbone, subsequent transformation into electrocompetent cells and estimation of theoretical complexity was done as described earlier5 (link).
3
Rat RNA-Seq Library Preparation
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The Ovation® Rat RNA-Seq System (NuGEN Technologies, San Carlos, CA) was used per manufacturer’s instructions to construct RNA-seq libraries. 100 ng of total RNA was used as input. First and second strands of cDNA were synthesized from total RNA (100 ng) spiked with 1 µl of 1:500 diluted ERCC ExFold RNA Spike-In Mix 2 (Life Technologies, Carlsbad, CA) at a final concentration of 1%. Following primer annealing and cDNA synthesis, end-repair, adaptor index ligation, and strand selection was conducted. Barcodes with unique indices was used per sample for multiplexing. Ribosomal RNA depletion was performed by using custom InDA-C primer mixture SS5 V8 for rat. Finally, libraries were amplified for 13 cycles (Mastercycler® pro, Eppendorf, Hamburg, Germany), and purified with Agencourt XP beads (Beckman Coulter, Indianapolis, IN).
4
Clonal Tracing of HSPC-Derived Cells
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The genetic barcodes were extracted from gDNA and amplified via PCR as described earlier [39 (link),40 (link)]. In brief, 200 ng of genomic DNA from PB, BM, or spleen was used in a single PCR reaction, using the oligos listed in Supplementary Table S2 . The obtained PCR products were purified using Agencourt XP-beads (Beckman Coulter, Brea, CA, USA) and quantified using the Qubit System (Life Technologies, Carlsbad, CA, USA). Finally, up to 65 PCR products were mixed in equal amounts to compose the libraries, which were subsequently sequenced on the Illumina MiSeq System (Illumina, San Diego, CA, USA) using single-end reads of 83 bps length. After demultiplexing, the NGS results were analyzed utilizing an in-house developed R-package [39 (link),70 (link)]. Barcode counts were corrected for the VCN (Supplementary Table S3 ), reflecting the reconstitution of the HSPC-derived clones. Subsequent measurements and clonal tracing within the same recipient were performed in animals from the last group (16–18 weeks) (Supplementary Figure S4 ). We used the R-Package “vegan” to calculate the Shannon index of our samples (https://cran.r-project.org/web/packages/vegan/index.html , last access 28 January 2022).
5
ChIP-seq Library Preparation for SOLiD Sequencing
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ChIP-seq libraries for all the samples were prepared from around 5 ng of DNA using fragment library construction kit from Life technologies for SOLiD sequencing. Briefly ChIPed DNA samples were end repaired and adapters were ligated using T4 DNA ligase. Ligated DNAs were subsequently amplified using adapter specific bar-coded primers for 15 cycles. DNA purification at every step was performed using Agencourt XP beads (Beckman Coulter). Library profiles were assessed using Agilent bioanalyzer 2100 high-sensitivity DNA kit. Equimolar amount of libraries were pooled and 50 bp reads were sequenced in-house using SOLiD 4.0 sequencer (Applied Biosystems).
6
Robust SOLiD Library Preparation for ChIP-Seq
7
Transcriptomic Analysis of Flavivirus
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5 μl of RNA was combined with 1 μl of 10 mM deoxynucleoside triphosphates (dNTPs), 1 μl of cDNA primer (ID_cDNAWNV_7374_Rev) (10 μM) and 3 μl of nuclease-free water. The 10 μl reaction volume was incubated for 5 minutes at 65°C and then placed on ice for 2 minutes. A reverse transcription reaction mixture containing 1μl of Superscript III RT enzyme, 1μl of RNaseOut, 2 μl dithiothreitol (DTT), 4 μl of 25 mM MgCl2, and 2 μl of 10x SSIII buffer was added to the previous reaction volume (20 μl total reaction volume) and incubated for 50 minutes at 50°C, followed by 5 minutes at 85°C. Reactions were chilled on ice, spun down and incubated for 20 minutes at 37 °C after an addition of 1μl of RNaseH. cDNA was purified using Agencourt xp beads (Beckman Coulter) at 1X concentration with elution into 12μl of nuclease-free water.
8
WNV amplicon library preparation
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Two steps were used for amplification of the target amplicon. 11 μl of cDNA was combined with 0.75 μl of 10 nM forward primer (R1_5’_WNV_for), 0.75 μl of 10 nM forward primer (5’_ID_Primer_Rev), and 12.5 μl of 2x KAPA HiFi HotStart mastermix (VWR). PCR conditions were 95°C for 3 min, 98°C for 20 s, 72°C for 45 s, 72°C for 1 min with 35 cycles. Samples were purified using Agencourt XP beads (Beckman Coulter) at 0.6X concentration and eluted in 20 μl of nuclease-free water.
9
cDNA Circularization and Purification
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To the cDNA, 3 μl 10× CircLigase Buffer (Epicentre), 1.5 μl 50 mM MnCl2, 1.5 μl 1 mM ATP, 0.5 μl 100 μM DNA adapter (oligonucleotide I; Supplementary Table S1 ), and 1 μl CircLigase I (Epicentre) were added. The reaction was incubated at 60°C for 2 h, then 80°C for 10 min to inactivate the ligase. The ligated DNA was EtOH precipitated with 20 mg glycogen as a carrier and dissolved in 20 μl of nuclease-free H2O. Then the cDNA was purified using 36 μl of Agencourt XP beads (Beckman Coulter), according to manufacturer's instructions and eluted with 20 μl TE buffer.
10
Rat RNA-Seq Library Preparation
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The Ovation® Rat RNA-Seq System 1–16 (NuGEN Technologies, San Carlos, CA) was used per manufacturer’s instructions to construct RNA-seq libraries. 100 ng of total RNA was used as input. First and second strands of cDNA were synthesized from total RNA (100 ng) spiked with 1 μl of 1:500 diluted ERCC ExFold RNA Spike-In Mix 2 (Life Technologies, Carlsbad, CA) at a final concentration of 1%. Following primer annealing and cDNA synthesis, end-repair, adaptor index ligation, and strand selection was conducted. Barcodes with unique indices was used per sample for multiplexing. Ribosomal RNA depletion was performed by using custom InDA-C primer mixture SS5 V8 for rat. Finally, libraries were amplified for 13 cycles (Mastercycler® pro, Eppendorf, Hamburg, Germany), and purified with Agencourt XP beads (Beckman Coulter, Indianapolis, IN).
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