Total RNA of each sample was used to prepare the miRNA sequencing library, which included the following steps: 1) 3'-adapter ligation with T4 RNA ligase 2 (truncated); 2) 5'-adapter ligation with T4 RNA ligase; 3) cDNA synthesis with an RT primer; 4) PCR amplification; and 5) extraction and purification of 120–140 bp PCR amplified fragments (corresponding to ~15–25 nt small RNAs) from polyacrylamide gels. An Agilent 2100 Bioanalyzer quantified the libraries, after which the samples were diluted to a final concentration of 8 pM and cluster generation was performed on the Illumina cBot using TruSeq Rapid SR cluster kit (#GD-402–4001, Illumina), following the manufacturer’s instructions. The DNA fragments in the libraries were denatured with 0.1M NaOH to generate single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ and finally sequenced for 36 cycles on Illumina HiSeq 2000 (Illumina, San Diego, CA, USA), according to the manufacturer’s instructions [41 (link)].
Truseq rapid sr cluster kit
Manufactured by Illumina
Sourced in United States
The TruSeq Rapid SR Cluster Kit is a laboratory equipment product from Illumina designed for the automated preparation of DNA libraries for sequencing on Illumina sequencing platforms. The kit provides the necessary reagents and consumables to cluster generation for single-read sequencing applications.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2000, by Illumina (11 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (8 mentions)
Truseq rapid sbs kit, by Illumina (7 mentions)
Nextseq 500, by Illumina (4 mentions)
Rt primers and amplification primers, by Illumina (2 mentions)
2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nanodrop nd 1000, by Thermo Fisher Scientific (1 mentions)
Flow cell, by Illumina (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Nextseq 500 sequencer, by Illumina (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Truseq mirna sample prep kit v2, by Illumina (1 mentions)
Hiseq 2000 system, by Illumina (1 mentions)
17 protocols using truseq rapid sr cluster kit
1
miRNA Sequencing Library Preparation
2
Pooled Blood PMN Transcriptome Profiling
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For primary blood PMN samples involving cell isolation, we used a pooling strategy involving equal mixing of total RNA samples derived from independent biological samples. Pooling of small RNA samples is effective in reducing data variability, and it reduces the number of replicates, hence lowering the cost for subsequent steps [64 (link)]. For each of the other samples, a unique total RNA specimen was analyzed.
The quality and the concentration of the total RNA samples were verified using a NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA) and gel separation (Supplementary Table S15 ). The sRNA-Seq libraries, containing RNA species between 8 and 30 nt in length were prepared as previously described [65 (link)]. Samples were diluted to a final concentration of 8 pM, denatured as single-stranded DNA, and cluster generation was performed on the Illumina cBot using a TruSeq Rapid SR cluster kit (GD-402-4001, Illumina, San Diego, CA, USA). Afterward, the clusters were sequenced for 51 cycles on Illumina HiSeq 2000 using TruSeq Rapid SBS Kits (FC-402-4002, Illumina, San Diego, CA, USA), as per the manufacturer’s instructions.
The quality and the concentration of the total RNA samples were verified using a NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA) and gel separation (
3
Illumina Sequencing Library Preparation
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The samples were diluted to a final concentration of 8 pM and cluster generation was performed on the Illumina cBot using TruSeq Rapid SR cluster kit (Illumina, Inc.), following the manufacturer's protocols. Sequencing was performed on an Illumina HiSeq 2000 using TruSeq Rapid SBS kits (Illumina, Inc.), according to the manufacturer's protocols. The DNA fragments in the libraries were denatured with 0.1 M NaOH to generate single-stranded DNA molecules, captured on flow cells (Illumina, Inc.), amplified in situ and finally sequenced for 36 cycles on Illumina HiSeq 2000.
4
miRNA Sequencing Library Preparation
5
miRNA Sequencing of PCMV-Infected Tissues
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The total RNA of PCMV-infected and uninfected lungs, liver, spleen, kidneys and thymus was isolated using Trizol reagent (Invitrogen, Waltham, MA, USA). After 3′- and 5′-adapter ligation with T4 RNA ligase, cDNA was synthesised using RT primer, and PCR products with a length of 120–140 bp were purified using polyacrylamide gel. The high-quality samples from PCMV-infected and uninfected groups were diluted to a concentration of 8 pM for cluster generation using TruSeq Rapid SR cluster kit (Illumina, San Diego, CA, USA), following the manufacturer’s instructions. miRNA high-throughput sequencing was performed using Illumina HiSeq 2000 (Illumina, San Diego, CA, USA) for 36 cycles.
6
miRNA Sequencing of Tfh Cells
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Total RNA samples were extracted from AR or WT mouse-derived Tfhs using TRIzol (Invitrogen, Shanghai, China). RNA samples were subject to agarose electrophoresis, NanoDrop ND-1000 (Nano Drop Inc., Wilmington, DE, USA) quantification, and then used to prepare miRNA sequencing library using NEB Multiplex Small RNA Library Prep Set for Illumina in five steps: 1) 3’-adapter ligation; 2) 5’-adapter ligation; 3) cDNA synthesis; 4) PCR amplification; and 5) extraction and purification of 135–155 bp PCR amplified fragments (corresponding to ~15–35 nt small RNAs). An Agilent 2100 Bioanalyzer (Agilent, Beijing, China) was used to quantify the libraries. The DNA fragments in the library were denatured with 0.1 M NaOH, captured on Illumina flow cells, amplified in situ, and sequenced for 50 cycles on Illumina NextSeq 500 sequencer (Illumina, San Diego, CA, USA) at Aksomics, Inc. (Shanghai, China). Sequencing reaction was performed using TruSeq Rapid SR Cluster Kit (#GD-402-4001, Illumina).
7
Small RNA-seq Profiling of Circulating miRNA
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Profiling of circulating miRNA was performed by small RNA-seq in Illumina NextSeq 500 platform as previously described [24 (link)]. Briefly, small RNA libraries were generated using the TruSeq® miRNA Sample Prep kit v2 (Illumina, San Diego, CA, USA). RNA template of each sample was sequentially ligated to 3′ and 5′ adapters, reverse transcribed, PCR amplified and selected on agarose gels by size of ~130–150 bp (correspond to ~15–35nt small RNAs). Then, PCR amplified fragments were eluted from gel pieces, purified and quantified by Agilent 2100 Bioanalyzer (Agilent, Santa Clara, CA, USA). The completed libraries were diluted to a final concentration of 8 pM and processed for cluster generation on the Illumina cBOT using TruSeq Rapid SR cluster kit (Illumina, San Diego, CA, USA). Sequencing was performed on Illumina NextSeq 500 platform using TruSeq Rapid SRB kits (Illumina, San Diego, CA, USA).
8
Illumina HiSeq 2000 Sequencing
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The library was denatured with 0.1 M NaOH to generate single-stranded DNA molecules and loaded onto channels of the flow cell at 8 pM concentration, amplified in situ using TruSeq Rapid SR Cluster Kit (Illumina, San Diego, CA, USA). Sequencing was carried out by running 100 cycles on Illumina HiSeq 2000 according to the manufacturer's instructions. The Agilent 2100 Bioanalyzer was used for accurate assessment of the quality and concentration of the sequencing library, while the size and concentration of each sample were determined after sequencing library preparation.
9
DNA Sequencing Using Illumina HiSeq 2000
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Samples were diluted to a final concentration of 8 pM, denatured as single-stranded DNA, and cluster generation was performed on the Illumina cBot using TruSeq Rapid SR cluster kit (GD-402-4001, Illumina, San Diego, CA, USA). Afterward, the clusters were sequenced for 51 cycles on Illumina HiSeq 2000 using TruSeq Rapid SBS Kits (FC-402-4002, Illumina, San Diego, CA, USA), as per the manufacturer’s instructions.
10
Profiling miRNA Expression in Kidney Injury
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Total RNA was extracted from the injured kidney tissue of mice following CIH exposure. The small RNA libraries were generated with the help of the Illumina TruSeq Rapid SR Cluster Kit. The purified cDNA libraries were utilized for cluster generation on an Illumina’s flow cell and then sequenced on Illumina NextSeq 500 according to the manufacturer’s instructions.
The miRNA deep sequencing data were analyzed by miRDB and miRBase. Volcano plot analysis was conducted using R software. Hierarchical clustering was performed using Cluster 3.0. GO analysis was used to categorize the function of DEmiRs. The KEGG pathway analysis was used to identify the main signaling pathways. The miRNA-seq experiment was employed in triplicate.
The miRNA deep sequencing data were analyzed by miRDB and miRBase. Volcano plot analysis was conducted using R software. Hierarchical clustering was performed using Cluster 3.0. GO analysis was used to categorize the function of DEmiRs. The KEGG pathway analysis was used to identify the main signaling pathways. The miRNA-seq experiment was employed in triplicate.
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