Six-hundred nanograms of 4C template was used for PCR amplification using Sigma-Aldrich long-template PCR system with two-step PCR system from Illumina. Bait-specific inverse primers conjugated to Illumina sequencing adaptors (primer sequences are in the Supplemental Table S3 ) were used in a first PCR reaction in a final volume of 50 µL with the following program: 2 min at 94°C followed by 20 cycles of 15 sec at 94°C, 1 min at 55°C, and 3 min at 68°C and a final extension of 7 min at 68°C. PCRs were performed in parallel reactions with 6 × 100 ng of template for each sample. PCR products were purified with the AMPure XP beads system (Beckman Coulter). Purified products were pooled and used as the template of a second PCR reaction with Nextera XT index kit version2 primers (FC-131-2004) in a final volume of 50 µL with the following program: 2 min at 94°C followed by 10 cycles of 15 sec at 94°C, 1 min at 55°C, and 3 min at 68°C and a final extension of 7 min at 68°C. PCR products were purified with the AMPure XP beads system (Beckman Coulter) and then sequenced on NextSeq 500 machines using single-end 75-bp read length.
Ampure xp beads system
Manufactured by Beckman Coulter
Sourced in United States
The AMPure XP beads system is a magnetic bead-based purification method used to selectively bind and purify nucleic acids, such as DNA and RNA, from solution. The system utilizes carboxylate-coated paramagnetic beads to capture and concentrate target molecules, allowing for efficient removal of unwanted components, like salts, enzymes, and other contaminants. This provides a simple and effective way to purify nucleic acids for a variety of downstream applications.
Automatically generated - may contain errors
Lab products found in correlation
Long template pcr system, by Illumina (2 mentions)
Miseq reagent kit v3 600 cycle, by Illumina (2 mentions)
Gentra puregene kit, by Qiagen (2 mentions)
Sequencing adaptor, by Illumina (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Nebnext adaptor oligonucleotides, by Illumina (1 mentions)
Neb universal pcr primer and index primer, by Illumina (1 mentions)
Poly t oligo attached magnetic beads, by Thermo Fisher Scientific (1 mentions)
Truseq pe cluster kit v3 cbot hs, by Illumina (1 mentions)
Hiseq 2000 platform, by Illumina (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Hiseq x10, by Illumina (1 mentions)
User enzyme, by New England Biolabs (1 mentions)
6 protocols using ampure xp beads system
1
Illumina Library Preparation from 4C Templates
2
PCR Amplification of 4C Template
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Six-hundred nanograms of 4C template was used for PCR amplification using Sigma-Aldrich long-template PCR system with bait-specific inverse primers conjugated to Illumina sequencing adaptors (primer sequences are in Supplemental Table S3 ) in a final volume of 50 µL in the following PCR program: 2 min at 94°C followed by 30 cycles of 15 sec at 94°C, 1 min at 55°C, and 3 min at 68°C and a final extension of 7 min at 68°C. PCR were performed in parallel reactions with 6 × 100 ng of template for each sample. PCR products were purified with the AMPure XP beads system (Beckman Coulter), and amplification profiles were analyzed by fragment analyzer and then sequenced on Illumina HiSeq 2000 machines using single-end 100-bp read length.
3
T Cell Receptor Sequencing from Genomic DNA
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DNA was isolated from cell lysate using the Gentra Puregene Kit (QIAGEN, cat# 159667). The quantity of DNA isolated and sequenced per sample is listed in Table S2 . Targeted PCR was used for amplification of TRB sequences from genomic DNA, using a cocktail of forward primers specific for framework region 2 (FR2) sequences of 23 TRBV subgroups (gene families), and 13 TRBJ region reverse primers adapted from the BIOMED2 primer series. Amplicons were purified using the Agencourt AMPure XP beads system (Beckman Coulter, cat# A63881). To generate the sequencing libraries, second-round PCRs were carried out using Nexter-aXT Index Primers S5XX and N7XX. Libraries were sequenced using an Illumina MiSeq in the Human Immunology Core Facility at the University of Pennsylvania. 2×300 bp paired end kits were used for all experiments (Illumina MiSeq Reagent Kit v3, 600 cycle, Illumina, cat# MS102-3003).
4
T Cell Receptor Sequencing from Genomic DNA
5
mRNA-seq Library Preparation Protocol
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mRNA was purified from total RNA using poly-T oligo-attached magnetic beads (Life Technologies, USA) and transcribed to cDNA using random oligonucleotides and M-MuLV Reverse Transcriptase (RNase H−) (TaKaRa, Dalian, China). NEBNext adaptor oligonucleotides (Illumia, USA) were ligated to 3′ ends of cDNA fragments. Then, 200-bp cDNA fragments were purified using the AMPure XP beads system (Beckman Coulter, USA). Ten cycles of PCR amplifications were performed to enrich cDNA fragments using the NEB Universal PCR primer and Index primer (Illumia, USA). The PCR products were purified using the AMPure XP beads system and quantified using the Agilent Bioanalyzer 2100 system. Finally, the four-coded samples were clustered by a cBot Cluster Generation System using the TruSeq PE Cluster Kit v3-cBot-HS (Illumia, USA), and then sequenced on an Illumina Hiseq 2000 platform.
6
Directional RNA-Seq Library Preparation
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The cDNA libraries were prepared by NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB, USA, #E7760S) following manufacturer’s recommendations. Briefly, the first strand cDNA was synthesized using random primers and M‐MuLV Reverse Transcriptase (RNaseH‐) after fragmentation of the RNAs. Then, the second strand cDNA was synthesized with DNA polymerase I and RNase H, and also, dUTP was introduced in this step. Subsequently, remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of 3ʹ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. 150–200 bp of cDNA fragments were enriched in the following size selection step by using AMPure XP beads system (Beckman Coulter, Beverly, USA). Then, the libraries were digested with 3 μL USER Enzyme (NEB, USA) at 37°C for 15 minutes. Then, preamplification was performed with Phusion High‐Fidelity DNA polymerase, and Index was introduced in this step. Finally, the PCR products were purified and cDNA library concentration was assessed using a Qubit® 2.0 fluorometer. The library was sequenced by Illumina HiSeq X-10 (Illumina Inc., San Diego, CA, USA) sequencer using a 2 × 150 bp paired-end pattern (PE150).
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