Total RNA was purified using a miRNAeasy micro kit (Qiagen, USA) and quantified as described previously (12 (link), 15 (link)). Purified total RNA (5 ng) was amplified following the Smart-seq2 protocol (16 (link)). cDNA was purified using AMPure XP beads (1:1 ratio, Beckman Coulter). From this step, 1 ng of cDNA was used to prepare a standard Nextera XT sequencing library (Nextera XT DNA sample preparation kit and index kit, Illumina). Samples were sequenced using a HiSeq2500 (Illumina) to obtain 50-bp single end reads. Both whole-transcriptome amplification and sequencing library preparations were performed in a 96-well format to reduce assay-to-assay variability. Quality control steps were included to determine total RNA quality and quantity, the optimal number of PCR pre-amplification cycles, and fragment size selection. Samples that failed quality control were eliminated from further downstream steps. Barcoded Illumina sequencing libraries (Nextera, Illumina) were generated utilizing the automated platform (Biomek FXp). Libraries were sequenced on the HiSeq2500 Illumina platform to obtain 50-bp single end reads (TruSeq® Rapid Kit, Illumina), generating a total of >700 million mapped reads (median of ~9 million mapped reads per sample).
Nextera xt dna sample preparation kit and index kit
Manufactured by Illumina
The Nextera XT DNA sample preparation kit and index kit are laboratory equipment used for the preparation of DNA samples for sequencing. The kit provides a streamlined workflow for library preparation, enabling efficient and consistent sample processing. The core function of the kit is to prepare DNA samples for next-generation sequencing analysis.
Automatically generated - may contain errors
Lab products found in correlation
Ampure xp bead, by Beckman Coulter (8 mentions)
Truseq rapid kit, by Illumina (5 mentions)
Mirneasy micro kit, by Qiagen (5 mentions)
Hiseq 2500, by Illumina (3 mentions)
Mirnaeasy micro kit, by Qiagen (2 mentions)
Biomek fxp, by Illumina (2 mentions)
Dsdna hs assay kit, by Thermo Fisher Scientific (1 mentions)
Hiseq 2500 system, by Illumina (1 mentions)
Platinum taq dna polymerase kit, by Thermo Fisher Scientific (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Hiseq sr cluster kit v4 cbot, by Illumina (1 mentions)
Hiseq sbs kit v4, by Illumina (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
10 protocols using nextera xt dna sample preparation kit and index kit
1
Single-cell RNA-seq Library Preparation and Sequencing
2
Transcriptome profiling of memory CD8 T cells
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RNA sequencing (RNA-seq) of memory CD8 T cells and CD8+/− MR1tet+ cells was performed as described previously (31 (link)). Briefly, total RNA was purified using an miRNeasy Micro Kit (QIAGEN) and quantified by quantitative PCR, as described previously (32 (link)). Purified total RNA (1–5 ng) was amplified following the Smart-Seq2 protocol (33 (link)). mRNA was captured using poly-dT oligonucleotides and directly reverse transcribed into full-length cDNA (amplified by PCR for 16 cycles). cDNA was purified using AMPure XP beads (Beckman Coulter). From this step, 1 ng of cDNA was used to prepare a standard Nextera XT sequencing library (Nextera XT DNA Sample Preparation Kit and Index Kit; Illumina). Whole-transcriptome amplification and sequencing library preparations were performed in a 96-well format to reduce assay-to-assay variability. Quality-control steps were included to determine total RNA quality and quantity, the optimal number of PCR preamplification cycles, and fragment size selection. Samples that failed quality control were eliminated from further downstream steps. Barcoded Illumina sequencing libraries (Nextera; Illumina) were generated using the automated platform (Biomek FXp). Libraries were sequenced on an HiSeq 2500 Illumina platform to obtain 50-bp single-end reads (TruSeq Rapid Kit; Illumina).
3
Quantification and Sequencing of Diluted cDNA
4
Mutation Validation Using PCR Amplicon Sequencing
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Validation of each mutation was performed in matched primary, secondary recipient, and constitutional DNA, using PCR amplicon deep sequencing. Primers were designed using Primer-BLAST (sequences available upon request). PCR was performed using Platinum Taq DNA Polymerase kit (Life Technologies) according to the manufacturer’s instructions and products were analyzed using gel electrophoresis. PCR amplicons were purified using AMPure XP beads (Beckman Coulter Inc., Brea, CA) and prepared for sequencing using the Nextera XT DNA Sample Preparation Kit and Index Kit (Illumina). 2 × 150 bp paired-end sequencing was performed using the Illumina NextSeq 500 (Illumina) and paired-end reads were aligned to mm9 using BWA (0.7.12).
5
Transcriptome profiling of memory CD8 T cells
6
Bulk RNA-seq Library Preparation
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RNA sequencing was performed as described previously [24] (link). Briefly, total RNA was purified using an miRNeasy Micro Kit (QIAGEN) and quantified by quantitative PCR, as described previously [25] (link). Purified total RNA (1–5ng) was amplified following the Smart-Seq2 protocol (16 cycles of cDNA amplification) [26] (link). cDNA was purified using AMPure XP beads (Beckman Coulter). From this step, 1 ng of cDNA was used to prepare a standard Nextera XT sequencing library (Nextera XT DNA sample preparation kit and index kit, Illumina). Whole-transcriptome amplification and sequencing library preparations were performed in a 96-well format to reduce assay-to-assay variability. Quality-control steps were included to determine total RNA quality and quantity, the optimal number of PCR preamplification cycles, and fragment size selection. Samples that failed quality control were eliminated from further downstream steps. Barcoded Illumina sequencing libraries (Nextera; Illumina) were generated using the automated platform (Biomek FXp). Libraries were sequenced on a NovaSeq 6000 Illumina platform to obtain 50-bp paired end reads (TruSeq Rapid kit; Illumina).
7
Single-cell RNA-seq of SLE Patients
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Cells from three SLE (IFNpos) patients were sorted into plates to generate sequencing libraries following the Smart-seq2 protocol. cDNAs were purified twice with AmPure XT beads (0.8×). From each sample, 0.4 ng of cDNA was used to generate a sequencing library using the Nextera XT DNA sample preparation kit and index kit from Illumina. Quality control steps were included after each step to eliminate samples with low quality from the downstream process. A total of 156 single-cell libraries passed all quality control criteria. The libraries were pooled, then sequenced on a HiSeq 2500 to obtain a minimum of 50,000 50-bp single-end reads. A detailed protocol has been previously published (Rosales et al. 2018 (link)).
8
Single-cell RNA-seq from Sorted Cells
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Total RNA was isolated from sorted cell populations using an miRNeasy Micro kit (Qiagen) and quantified. Three nanograms of total RNA were used to generate cDNA following the Smart-seq2 protocol. cDNA was purified using AMPure XP beads (0.8×, Beckman Coulter). Next, 1 ng of cDNA from each sample was used to generate a sequencing library (Nextera XT DNA sample preparation kit and index kit, Illumina). The libraries were pooled and sequenced on a HiSeq 2500 (Illumina) to obtain 50-bp single-end reads. Both full-transcriptome amplification and sequencing library preparations were performed in a 96-well format to reduce assay-to-assay variability. Quality control steps were included after each step to eliminate samples with low quality from the downstream process. A detailed protocol has been previously published (Rosales et al. 2018 (link)). Libraries were sequenced on a HiSeq 2500 Illumina to obtain a minimum of 10 million 50-bp single-end reads (HiSeq SR Cluster kit v4 cBot, HiSeq SBS kit v4).
9
Streamlined RNA-seq Library Preparation
10
Bulk RNA-seq of Purified Cell Populations
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Bulk RNA-seq of purified cells was performed described previously 94 . Total RNA extraction was performed using a miRNAeasy micro kit (Qiagen, USA). Following quantification, RNA was amplified using the smart-seq2 protocol. DNA was purified using AMPure XP beads (1:1.1 ratio, Beckman Coulter). 1 ng of purified cDNA was used to prepare a standard Nextera XT sequencing library (Nextera XT DNA sample preparation kit and index kit, Illumina). Sequencing was performed using HiSeq2500 (Illumina) to obtain 50bp single-end reads (Supplementary Table 2). Quality control steps were included to determine total RNA quality and quantity, optimal number of PCR pre-amplification cycles, and cDNA fragment size. Samples that failed quality control were eliminated from further downstream analysis.
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