Agencourt xp dna beads

Manufactured by Beckman Coulter

Agencourt XP DNA beads are a magnetic bead-based solution for the purification and recovery of nucleic acids. They provide a simple and efficient method for the extraction and isolation of DNA from various sample types.

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Lab products found in correlation

Kapa hifi hotstart readymix, by Roche (2 mentions) Maxima h minus reverse transcriptase, by Thermo Fisher Scientific (2 mentions) Hiseq x machine, by Illumina (2 mentions) Triton x 100, by Merck Group (2 mentions) Dntp mix, by Thermo Fisher Scientific (2 mentions) 2100 bioanalyzer, by Agilent Technologies (1 mentions) Qubit dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions) Hiseq 4000 sequencer, by Illumina (1 mentions) Oligo dt primer, by Merck Group (1 mentions) Qubit hsdna kits, by Thermo Fisher Scientific (1 mentions) Nextseq 500 instrument, by Illumina (1 mentions) Maxima reverse transcriptase, by Thermo Fisher Scientific (1 mentions) Nextera xt, by Illumina (1 mentions) High sensitivity dna chip, by Agilent Technologies (1 mentions) Rnase inhibitor, by Takara Bio (1 mentions) Oligo dt primer, by Qiagen (1 mentions)

Close spelling variants of this product

Agencourt XP beads (18 citations) Agencourt AMPure XP DNA purification beads (10 citations) 0.8× XP DNA beads (2 citations)

6 protocols using agencourt xp dna beads

Single-cell Transcriptome Amplification and Sequencing

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5–15 metabolically active cells were retrieved and pooled together into low bind tube (Axygen) with 12.5 μl lysis buffer, which contained 10X Reaction buffer, ERCC (1:10⁶, Invitrogen), 3′ SMART-Seq CDS Primer IIA, RNase Inhibitor plus DEPC-treated water. Single-cell transcriptome amplifications were performed using SMART-Seq v4 Ultra Low RNA Kit as described in the protocol for the kit (Clotech). The amplified cDNA products were purified with 0.8X Agencourt XP DNA beads (Beckman Coulter). The concentration of purified cDNA was quantified with Qubit dsDNA HS Assay Kit (Invitrogen), and libraries were then constructed with the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme Biotech) and quantified by a 2100 Bioanalyzer (Agilent Technologies). Libraries were analyzed by an Illumina HiSeq X Ten sequencer with 150 bp pair-end reads (Genewiz, Suzhou, China).

Li Z., Wang Z., Tang Y., Lu X., Chen J., Dong Y., Wu B., Wang C., Yang L., Guo Z., Xue M., Lu S., Wei W, & Shi Q. (2019). Liquid biopsy-based single-cell metabolic phenotyping of lung cancer patients for informative diagnostics. Nature Communications, 10, 3856.

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Single-Cell RNA Sequencing with Modified Smart-seq2

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Libraries for isolated single cells were generated by the Smart‐seq2 method¹⁶ with the following modifications: RNA was reverse transcribed with Maxima H Minus Reverse Transcriptase (Thermo Fisher Scientific, MA, Cat: 00724792), and whole transcriptome was amplified using KAPA HiFi Hot Start Ready Mix (KAPA Biosystems, MA, Cat: KE2502). cDNA library was purified using Agencourt XP DNA beads (Beckman Coulter, CA, Cat: A63852) and quantified with a high sensitivity dsDNA Quant Kit (Life Technologies, CA, Cat: Q32854). It is worth mentioning that full length cDNA libraries were tagmented, and then only 3′ end sequence (500‐1000 bp) was amplified and enriched for sequencing on an Illumina HiSeqX machine, which is different from the traditional Smart‐seq2 method of full tagmented‐libraries sequencing.

Ruan H., Zhou Y., Shen J., Zhai Y., Xu Y., Pi L., Huang R., Chen K., Li X., Ma W., Wu Z., Deng X., Wang X., Zhang C, & Guan M. (2020). Circulating tumor cell characterization of lung cancer brain metastases in the cerebrospinal fluid through single‐cell transcriptome analysis. Clinical and Translational Medicine, 10(8), e246.

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Comprehensive Single-Cell Transcriptome Analysis

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One thousand cells of different subtypes including naïve and T_EM CD8+ T cells from PBMC, tumor-reactive CD8+ T cells, and two clusters of tumor bystander CD8+ T cells were enriched by gating 7AAD−CD3+CD8+CD45RO−CD45RA+CCR7+, 7AAD−CD3+CD8+CD45RO+CD45RA−CCR7−, 7AAD−CD3+CD8+CD103+CD39+, 7AAD−CD3+CD8+CD103+CD39−, and 7AAD−CD3+CD8+CD103−CD39−, respectively, and sorted into 0.2 mL tubes (Axygen) chilled to 4 °C, prepared with lysis buffer with 1 μL 10 mM dNTP mix (Invitrogen), 1 μL 10 μM Oligo dT primer, 1.9 μL 1% Triton X-100 (Sigma), and 0.1 μL 40 U μL⁻¹ RNase Inhibitor (Takara).
Transcriptome amplifications were performed according to Smart-Seq2 protocol [44 (link)] with modification of reagent amount and PCR cycle numbers. The amplified cDNA products were purified with Agencourt XP DNA beads (Beckman), and the concentration of each sample was quantified by Qubit HsDNA kits (Invitrogen). Libraries were constructed and amplified using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme Biotech). The libraries were then purified with Agencourt XP DNA beads and analyzed by fragment analyzer for quality assessment. Purified libraries were analyzed by an Illumina Hiseq 4000 sequencer with 150-bp pair-end reads.

Yang R., Cheng S., Luo N., Gao R., Yu K., Kang B., Wang L., Zhang Q., Fang Q., Zhang L., Li C., He A., Hu X., Peng J., Ren X, & Zhang Z. (2019). Distinct epigenetic features of tumor-reactive CD8+ T cells in colorectal cancer patients revealed by genome-wide DNA methylation analysis. Genome Biology, 21, 2.

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Plate-based scRNA-seq for Enriched Malignant Cells

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Plate-based scRNA-seq following FACS enrichment was used to strongly enrich for malignant cells. This approach complements efforts using droplet-based method (below) where the non-malignant compartment makes up the vast majority of cells, but also includes a relatively small portion of cancer cells but with more shallow data. To balance both approaches, we used both to complement different types of analyses. For plate-based sc-RNA-seq, we performed Whole Transcriptome Amplification (WTA) with a modified Smart-seq2 protocol, as described previously ^{15 (link),16} with Maxima Reverse Transcriptase (Life Technologies) instead of Superscript II. Next, WTA products were cleaned with Agencourt XP DNA beads and 70% ethanol (Beckman Coulter, Brea, CA) and Illumina sequencing libraries were prepared using Nextera XT (Illumina, San Diego, CA), as previously described¹⁶. The 96 samples of a multiwell plate were pooled, and cleaned with two 0.8X DNA SPRIs (Beckman Coulter). Library quality was assessed with a high sensitivity DNA chip (Agilent) and quantified with a high sensitivity dsDNA Quant Kit (Life Technologies). Barcoded single cell transcriptome libraries were sequenced with 38bp paired end reads on an Illumina NextSeq 500 Instrument.

Izar B., Tirosh I., Stover E.H., Wakiro I., Cuoco M.S., Alter I., Rodman C., Leeson R., Su M.J., Shah P., Iwanicki M., Walker S.R., Kanodia A., Melms J.C., Mei S., Lin J.R., Porter C.B., Slyper M., Waldman J., Jerby-Arnon L., Ashenberg O., Brinker T.J., Mills C., Rogava M., Vigneau S., Sorger P.K., Garraway L.A., Konstantinopoulos P.A., Liu J.F., Matulonis U., Johnson B.E., Rozenblatt-Rosen O., Rotem A, & Regev A. (2020). A single-cell landscape of high-grade serous ovarian cancer. Nature medicine, 26(8), 1271-1279.

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Single-cell RNA-seq of CSF Leukocytes

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Patient CSF samples (P: P1-P7) were diagnosed through cytopathology (Figure S1A) and 3 mL samples were remained for fluorescence-activated cell sorting (FACS). P1-P6 CSF cells sorted were CD45 (Catalog number Cat#560973, BD Biosciences) and CD19 (Cat#564456, BD Biosciences) positive, and had a larger cell diameter, showing greater forward scatter height (FSC-H) than the normal CSF leukocytes (Figure S1B). Whereas P7 CSF cells was only sorted based on CD45 positive. Live candidate CSF cells (CellTrace Calcein Blue AM+, Cat#C34853, Life Technologies, CA) were sorted into pre-prepared 96-well plates by FACS for SMART-seq2 scRNA-seq (Table S1 and Figure S1B). All antibody and labeling dye were used according to manufacturer recommendations. The construction of SMART-seq2 library was performed as the following modifications (Ruan et al., 2020 (link)): (1) RNA was reverse transcribed and amplified using Maxima H Minus Reverse Transcriptase (Cat#00724792, Thermo Fisher Scientific, MA) and KAPA HiFi Hot Start Ready Mix (Cat#KE2502, KAPA Biosystems, MA), (2) cDNA library was purified and quantified using Agencourt XP DNA beads (Cat#A63852, Beckman Coulter, CA) and a high sensitivity dsDNA Quant Kit (Cat#Q32854, Life Technologies, CA), (3) full length cDNA libraries were tagmented and only 3' end sequence (500-1000 bp) was enriched for sequencing on an Illumina HiSeqX machine.

Ruan H., Wang Z., Zhai Y., Xu Y., Pi L., Zheng J., Zhou Y., Zhang C., Huang R., Chen K., Li X., Ma W., Wu Z., Shen J., Deng X., Zhang C, & Guan M. (2021). Single-cell transcriptome analysis of diffuse large B cells in cerebrospinal fluid of central nervous system lymphoma. iScience, 24(9), 102972.

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Neoantigen-reactive TCR Identification

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A modified Smart-seq2 (Picelli et al., ) based method was used to identify neoantigen-reactive TCR sequences. First, T cells that upregulated an activation marker (4-1BB or OX40) upon co-culture with mutated TMGs or peptides were FACS sorted into wells of 96-well plate with lysis buffer, which contained 1 μl 10 mM dNTP mix (ThermoFisher Scientific), 1 μl 10 μM Oligo dT primer (Qiagen), 1.9 μl 1% Triton X-100 (Sigma-Aldrich) plus 0.1 μl 40 U/μl RNase Inhibitor (Takara). Single cell transcriptome amplifications were performed following the Smart-seq2 protocol with a modified TSO primer. TCR-alpha and -beta chains were further amplified with TCR specific primers. The amplified TCR cDNA products were purified with 0.7x Agencourt XP DNA beads (Beckman) twice and quantified with Qubit ) methods from RNA-Seq data.

Zheng C., Fass J.N., Shih Y.P., Gunderson A.J., Sanjuan Silva N., Huang H., Bernard B.M., Rajamanickam V., Slagel J., Bifulco C.B., Piening B., Newell P.H.A., Hansen P.D, & Tran E. (2022). Transcriptomic profiles of neoantigen-reactive T cells in human gastrointestinal cancers. Cancer cell, 40(4).

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