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Ampure xp magnetic beads

Manufactured by Illumina
Sourced in United States

AMPure XP magnetic beads are a nucleic acid purification reagent used for the purification of DNA, RNA, and other biomolecules. The beads selectively bind to the target molecules, allowing for the removal of contaminants and impurities through a series of washing steps. The purified samples can then be eluted from the beads for further downstream processing and analysis.

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10 protocols using ampure xp magnetic beads

1

Murine MLV Integration Site Sequencing

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Integration site sequencing was done as described previously (103 (link)). Briefly, mice were injected with WT MLV or W390A MLV. At 3 and 5 weeks postinfection or at a later stage of the disease (12 to 14 weeks postinfection), mice were sacrificed, and spleens were collected. Genomic DNA was extracted using the GenElute mammalian genomic DNA miniprep kit (Sigma). Sonication with the Covaris M220 instrument was used to shear genomic DNA randomly, and DNA linkers were ligated to the sheared DNA. Integration sites were amplified by nested PCR using iProof high-fidelity kit polymerase (Bio-Rad). Two sets of oligonucleotides that bind to the MLV LTR and the linker were used for PCR (103 (link)) (sequences are listed in Table 3), whereby Illumina sequencing adapters were linked to the second oligonucleotide. PCR products were purified using AMPure XP magnetic beads and sequenced using Illumina MiSeq (Illumina Inc., San Diego, CA, USA) paired-end reads with 300 cycles by the Leuven Genomics Core (KU Leuven). The relative abundance of each unique integration site was calculated according to the following formula: relative abundance = (abundance of integration sites/total abundance) × 100.
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2

Mouse TCR Library Preparation

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For TCR library preparation, we used the commercially available iRepertoire platform (iRepertoire, Huntsville, AL, USA) for nested amplicon arm-PCR of the CDR3 of the mouse TCR β-chains and addition of adaptors for Illumina platform sequencing. Reverse transcription of 300–500 ng of RNA was conducted with a One-Step reverse transcription and amplification kit (Qiagen, Germantown, MD, USA) according to the manufacturer’s protocol. The PCR product was purified using Ampure XP magnetic beads (Agencourt/Beckman Coulter, Brea, CA, USA), and secondary amplification of the resulting product was performed (GoTaq PCR Kit, Promega, Madison, WI), allowing addition of Illumina adapter sequences (manufacturer’s protocol). Libraries were purified with Ampure XP magnetic beads and sequenced using Illumina MiSeq 150 nt paired-end read-length. The TCR CDR3 sequences were extracted from the raw sequencing data by iRepertoire.
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3

Comprehensive Microbiome Profiling Protocol

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Cecal/fecal DNA was extracted using the E.Z.N.A stool DNA kit according to manufacturer’s instructions. For amplicon sequencing, the V4 region of the bacterial 16S gene was amplified from 10–50ng of DNA using KOD hot start DNA polymerase and barcoded primers (Caporaso et al., 2011 (link)). Libraries were size-selected with AMPure XP magnetic beads and sequenced on the Illumina MiSeq platform using the V3 600 cycle kit. Libraries for whole genome shotgun sequencing were prepared with the Nextera XT kit according to manufacturer’s protocol and sequenced on a HiSeq platform (paired-end 100bp at a depth of 15–20 million reads/sample) by the Integrated Genomics Operation (iGO) core facility at MSKCC.
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4

Murine MLV Integration Site Sequencing

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Integration site sequencing was done as described previously (103 (link)). Briefly, mice were injected with WT MLV or W390A MLV. At 3 and 5 weeks postinfection or at a later stage of the disease (12 to 14 weeks postinfection), mice were sacrificed, and spleens were collected. Genomic DNA was extracted using the GenElute mammalian genomic DNA miniprep kit (Sigma). Sonication with the Covaris M220 instrument was used to shear genomic DNA randomly, and DNA linkers were ligated to the sheared DNA. Integration sites were amplified by nested PCR using iProof high-fidelity kit polymerase (Bio-Rad). Two sets of oligonucleotides that bind to the MLV LTR and the linker were used for PCR (103 (link)) (sequences are listed in Table 3), whereby Illumina sequencing adapters were linked to the second oligonucleotide. PCR products were purified using AMPure XP magnetic beads and sequenced using Illumina MiSeq (Illumina Inc., San Diego, CA, USA) paired-end reads with 300 cycles by the Leuven Genomics Core (KU Leuven). The relative abundance of each unique integration site was calculated according to the following formula: relative abundance = (abundance of integration sites/total abundance) × 100.
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5

Sequencing of mtDNA from MWCNT-exposed BECs

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Purified mtDNA from MWCNT-exposed BECs were sequenced for the appearance of heteroplasmies and indel mutations compared to the sequences of vehicle-treated control cultures. Tagmentation of mtDNA (combined short and long fragments) was carried out using a Nextera DNA Library Prep kit (Illumina) according to the manufacturer’s instructions. DNA samples were added to a 96-well thermal cycler plate with Amplicon Tagment Mix and Tagment DNA buffer from the kit and heated at 55°C for 5 minutes. Libraries were then amplified with indexed adapters and Nextera PCR Master Mix, first denaturing at 72°C and then running 15 cycles of 95°C for 10 seconds, 55°C for 30 seconds, and 72°C for 30 seconds. AMPure XP magnetic beads (Illumina) were used to purify the resulting mtDNA libraries, according to manufacturer instructions. AMPure beads were added to each sample and allowed to incubate at room temperature for 7 minutes on a magnetic plate to pellet the DNA/beads. Supernatants were discarded and the bead pellets were washed twice with 80% ethanol, then allowed to air dry completely (approximately 10 minutes). Purified DNA was resuspended in AMPure Resuspension Buffer and the DNA concentration/quality was verified by QuBit High-Sensitivity Analysis (Thermo-Fisher) and the Agilent Bioanalyzer.
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6

16S rRNA Gene Sequencing of Mouse and Human Samples

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DNA was amplified using Kapa-HiFi Hotstart (KK2502, Kapa Biosystems) using primers to 16S-V4 regions (V4-515F - TCGTCGGCAGCGTCAGATGTGTATAAGAGACAGGTGCCAGCMGCCGCGGTAA, V4-806R - GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAGGGACTACHVGGGTWTCTAAT) on a BioRad CFX 384 real-time PCR instrument with four serial 10-fold dilutions of extracted DNA template. Individual sample dilutions in the exponential phase were selected using an OpenTrons OT2 for subsequent indexing PCR using a dual GoLay error-correcting index primers (37 (link)). DNA concentration was measured using a PicoGreen assay (P7589, Life Technologies, South San Francisco, CA, USA), and samples were pooled at equimolar concentrations. Agencourt AMPure XP magnetic beads were used to purify the pooled PCR product, and the samples were subsequently sequencing on an Illumina MiSeq using 15% PhiX spiked in for sequencing. Mouse samples were amplified using V4 primers as previously described (38 (link)). All sequencing was paired, with human 16S as 270 bp and mouse 16S as 150 bp fragments. Amplicon reactions were pooled at equimolar concentrations and purified using the Agencourt AMPure XP magnetic beads. The pooled library was loaded onto the Illumina NextSeq 550 platform using 40% PhiX spiked in for sequencing.
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7

Illumina Sequencing of Evolutionary Samples

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Illumina libraries were prepared for each of three samples (G0, G8, and G16 from cage 1:3B) using the NEXTFLEX PCR‐free library preparation kit and NEXTFLEX Unique Dual Index Barcodes (BIOO Scientific) following the manufacturer’s instructions. The input amount of DNA was 500 ng. The ends of the DNA were repaired and adenylated. The reaction was cleaned using AMPure XP magnetic beads and Illumina barcoded adapters were ligated onto the blunt-end adenylated product. The adapter-ligated product was cleaned using AMPure XP beads. DNA quantity was measured by Qubit DNA HS assay and the fragment size assessed by Agilent Bioanalyzer 2100 DNA HS chip assay at the genomics facility of the University of Utah (GNomEx) where the libraries were sequenced on the Illumina NovaSeq with the SP flowcell 2 × 250 paired end.
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8

Murine TCR beta sequencing protocol

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TCR sequencing was performed using the Mouse TCR beta, Illumina, V-C genes kit from iRepertoire, Huntsville, AL. In brief, 500 ng of RNA was reverse transcribed using a One-Step reverse transcription and amplification kit (Qiagen). The PCR product was purified using Ampure XP magnetic beads (Agencourt), and amplified again (TopTaq PCR Kit, Qiagen), to add adaptor sequences. Libraries were purified with Ampure XP magnetic beads and sequenced using Illumina MiSeq 150 nt paired-end read-length. The TCR CDR3 sequences were extracted from the raw sequencing data by iRepertoire. Briefly, raw paired-end fastq files were first demultiplexed based on barcode and merged reads were mapped using a Smith-Waterman algorithm to germline V, D, J and C reference sequences from the IMGT web site (http://www.imgt.org). To define the CDR3 region, the position of CDR3 boundaries of reference sequences from the IMGT database were migrated onto reads through mapping results and the resulting CDR3 regions were extracted and translated into amino acids. Reading frames not containing a stop codon were filtered and error-corrected using iRepertoire proprietary SMART algorithm.
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9

High-throughput TCR profiling of tumor-infiltrating Tregs

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CD25+FoxP3(GFP)+CD4+ Tregs were sorted into RL buffer (Norgen, 51800) from the omenta and spleens of 4 naïve and 4 day 15 EG7.1.15 tumor-bearing FoxP3-GFP-DTR mice. Sorted cells were not pooled, thus each sample represents a Treg population from an organ of an individual mouse (Supplemental Table 2). Total RNA was purified using Single Cell RNA purification kit (Norgen) according to the manufacturer’s instructions. TCR library was prepared using iRepertoire arm-PCR platform (iRepertoire) as previously described (31 (link)). Briefly, reverse transcription of RNA was conducted with OneStep RT-PCR kit (Qiagen, 210212) according to the manufacturer’s protocol. The PCR product was purified using Ampure XP magnetic beads (Agencourt, A63880), and secondary amplification of the resulting product was performed (GoTaq PCR Kit, Promega, M7660), allowing addition of Illumina adapter sequences. Finally, libraries were purified with Ampure XP magnetic beads and sequenced using Illumina MiSeq 250 nt paired-end read-length. The TCR CDR3 sequences were extracted from the raw sequencing data, wherein fastq files were first demultiplexed and reads were then mapped to germline V, D, J and C reference sequences from the IMGT using iRepertoire bioinformatics tools.
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10

MLV Integration Site Sequencing

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Integration site sequencing was done as described (58) . Briefly, mice were injected with WT MLV or W390A MLV. After 3 and 5 weeks post/infection or at a later stage of the disease (12 -14 weeks post-infection), mice were sacrificed, and spleens were collected. Genomic DNA was extracted using the GeneElute Mammalian Genomic DNA Miniprep Kit (Sigma). Sonication with the Covaris M220 was used to shear genomic DNA randomly and DNA linkers were ligated to the sheared DNA. Integration sites were amplified by nested PCR using the iProof High-Fidelity kit polymerase (BioRad). Two sets of primers were used in the PCR (sequences are listed in Table 1, indicated as MLV IN III and MLV IN IV), whereby Illumina sequencing adapters were linked to the second set of primers. PCR products were purified by AMPure XP magnetic beads and sequenced by Illumina Miseq (Illumina Inc., San Diego, CA, USA) paired-end 300 cycles by the Leuven Genomics Core (KU Leuven).
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