The library construction and sequencing of miRNA were conducted by OE Biotech Co., Ltd. miRNAs of MSC-exos were extracted by exoEasy Maxi Kit (QIAGEN) and quantified by Qubit 2.0 (Life Technologies). The integrity was confirmed by Agilent 2100 TapeStation (Agilent Technologies). A total of 50 ng exosomal RNA from each sample was used for small RNA library construction. Small RNA sequencing was conducted by HiSeq 2500 (Illumina) with reads lengths from 15 to 41 bp.
Agilent 2100 tapestation
Manufactured by Agilent Technologies
Sourced in United States, Canada
The Agilent 2100 TapeStation is a laboratory instrument designed for automated electrophoresis analysis of DNA, RNA, and protein samples. It provides rapid, high-resolution analysis of sample quality and concentration.
Automatically generated - may contain errors
Lab products found in correlation
Hiseq 2500, by Illumina (5 mentions)
Trizol reagent, by Thermo Fisher Scientific (5 mentions)
Qubit 2, by Thermo Fisher Scientific (3 mentions)
Exoeasy maxi kit, by Qiagen (3 mentions)
Hiscansq, by Illumina (3 mentions)
Truseq rna sample prep kit, by Illumina (3 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Rneasy kit, by Qiagen (2 mentions)
Nanodrop 1000, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions)
Nextseq 500 system, by Illumina (1 mentions)
Kapa mrna hyperprep kit, by Roche (1 mentions)
Hiseq x system, by Illumina (1 mentions)
Agencourt rnaclean xp beads, by Beckman Coulter (1 mentions)
Stranded mrna prep kit, by Illumina (1 mentions)
D1000 screentape, by Agilent Technologies (1 mentions)
Novaseq 6000, by Illumina (1 mentions)
Casava, by Illumina (1 mentions)
17 protocols using agilent 2100 tapestation
1
Exosomal miRNA Profiling by RNA-Seq
2
RNA-seq Analysis of NRVMs
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NRVMs were collected at 48 h after infection with adenoviruses for lacZ, Nrf1, and Nrf2. RNA was extracted using the RNeasy Mini Kit (QIAGEN, 74104) according to the manufacturer’s protocol. An Agilent 2100 TapeStation was used for RNA quality analysis. Stranded mRNA-seq libraries were generated using the KAPA mRNA HyperPrep Kit (Roche, KK8581) following the manufacturer’s protocol. Sequencing was performed on an Illumina NextSeq 500 system using a 75-bp, high-output sequencing kit for single-end sequencing.
For data analysis, quality control of RNA-seq data was performed using the FastQC Tool (Version 0.11.4). Sequencing reads were aligned to rat Rnor 6.0 reference genome using HiSAT2 (Version 2.0.4) with default settings and -rna-strandness F55 (link). After removal of duplicate reads using Samtools v0.1.1856 (link), aligned reads were counted using featurecount (Version 1.6.0) per gene ID57 (link). Differential gene expression analysis was performed with the R package edgeR (Version 3.20.5) using the GLM approach58 (link). For each comparison, genes with more than 1 count per million (CPM) in at least three samples were considered as expressed and were used for calculating the normalization factor. Cutoff values of absolute fold-change >1.5 and false discovery rate <0.05 were used to select DEGs between sample group comparisons.
For data analysis, quality control of RNA-seq data was performed using the FastQC Tool (Version 0.11.4). Sequencing reads were aligned to rat Rnor 6.0 reference genome using HiSAT2 (Version 2.0.4) with default settings and -rna-strandness F55 (link). After removal of duplicate reads using Samtools v0.1.1856 (link), aligned reads were counted using featurecount (Version 1.6.0) per gene ID57 (link). Differential gene expression analysis was performed with the R package edgeR (Version 3.20.5) using the GLM approach58 (link). For each comparison, genes with more than 1 count per million (CPM) in at least three samples were considered as expressed and were used for calculating the normalization factor. Cutoff values of absolute fold-change >1.5 and false discovery rate <0.05 were used to select DEGs between sample group comparisons.
3
Ribosomal RNA Depletion and Strand-Specific RNA-Seq
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Ribosomal RNA (rRNA) in the samples was depleted using the NEBNext rRNA Depletion Kit (Bacteria) (NEB) and cleaned up using Agencourt RNAClean XP Beads (Beckman Coulter). Strand-specific sequencing libraries were prepared using an Illumina Stranded mRNA Prep Kit (Illumina) following the manufacturer’s instructions with minor modifications. rRNA-depleted samples in 4.25 µL of nuclease-free water were mixed directly with 4.25 µL of the Elution, Prime, Fragment 3HC Mix (EPH3) solution, and fragmented/denatured at 94°C for 5 minutes. The remaining steps were performed according to the manufacturer’s instructions using half volumes of reagents, except the final cDNA elution step. The sizes and concentrations of the libraries were determined on an Agilent 2100 Tapestation using a D1000 ScreenTape. Libraries were pooled and sequenced on an Illumina Hiseq X system.
4
Universal CUT&Tag Library Preparation
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The CUT&Tag experiments, including the construction of DNA library, were carried out according to the protocol of hyperactive Universal CUT&Tag Assay Kit (TD903‐01/02, vazyme). Briefly, cells were harvested and counted (100,000 cells). The genomic DNA (gDNA) samples were eluted using 26 μL of Millipore water, quantified with the Onedrop system, and then made ready for library amplification. The amplification cycles were 16 according to the index of Illumina (Vazyme#TD202‐01) and the products were purified by 1.2× magnetic beads (Vazyme#N411‐01), and the final library elution volume was 22 μL. The cDNA libraries underwent sequencing on the Illumina Novaseq 6000, employing 150‐bp paired ends and delegated to Novogene Biotech Co., Ltd. (Beijing China) after the size was determined by the Agilent 2100 Tape Station analysis. Detailed analysed results were listed in the final report of biological information analysis (Additional files 3 : X101SC21061883‐Z01‐J001‐B1‐36)
5
Illumina-Based RNA Sequencing Analysis of Gene Expression
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Total RNA was prepared using a Qiagen RNeasy Plus Mini Kit and processed by the MUSC Genomics core for 101-bp, paired-end RNA sequencing on an Illumina HiScanSQ. RNA integrity was verified on an Agilent 2100 TapeStation (Agilent Technologies, Palo Alto, CA). 100–200 ng of total RNA was used to prepare RNA-Seq libraries using the TruSeq RNA Sample Prep kit following the protocol described by the manufacturer (Illumina, San Diego, CA). Sequencing was performed on an Illumina HiScanSQ. Samples were demultiplexed using CASAVA (Illumina, San Diego, CA). Fastq files were used to map reads to the human genome (hg19, UCSC) utilizing Tophat2 with default settings. Bam files were imported into Partek Genomics Suite (St. Louis, MO) for analysis. Samples were filtered for ≥ 10 reads. Differential gene expression was performed by comparison of biological duplicate samples in Genomics Suite using advanced analysis of variance (ANOVA). Differential gene express values were then filtered by ± 1.5 fold change and by unadjusted P ≤ .05.
6
RNA-seq Library Preparation and Sequencing
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The RNA-seq library was generated using VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina according to the manufacturer’s protocol. The quality of each library was controlled using an Agilent 2100 TapeStation (Agilent). The libraries were pooled and sequenced on an Illumina NovaSeq 6000 platform following to the manufacturer’s protocol and finally 150bp paired-end reads were generated.
7
RNA Extraction and Quality Assessment
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According to the manufacturer’s instructions, the total RNA was extracted from each WB sample employing TRIzol reagent (Invitrogen, Carlsbad, CA, United States). RNA purity was determined by using a spectrophotometer (NanoDrop-1000, Thermo Fisher Scientific) with OD260/OD280 readings (10mM Tris, pH 7.5) between 1.8 and 2.1. The temperature was maintained at −80°C, while the RNA quality was measured by the Agilent 2,100 TapeStation (Agilent Technologies, United States) Biochip Analysis System. Eventually, samples were selected with RNA integrity values > 7.0 to construct cDNA libraries. The RNA-seq bioinformatics pipeline is illustrated in Figure 1A , and the workflow for this study is schematically shown in Figure 1B .
8
RNA Extraction and Quality Assessment
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Total RNA extracts from each sample were obtained with TRIzol reagent, according to the manufacturer's instructions (Thermo Fisher Scientific, Inc.). The total RNA quality was analyzed using agarose electrophoresis (1% gels). The A260/A280 ratio was determined using a NanoDrop spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.). RNA integrity was assessed by Agilent 2100 TapeStation analysis (Agilent Technologies, Santa Clara, CA, USA). An A260/A280 ratio between 1.8 and 2.0 and an RNA integrity number >7 were considered acceptable parameters for RNA integrity. RNA sequencing was performed on an Illumina X-ten RNA-Seq sequence production system (Illumina, Inc., San Diego, CA, USA).
9
Transcriptome Profiling of Plant Varieties
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First, the total RNA of 60 samples (2 varieties × (control + (3 treatments × 3 time points)) × 3 biological replicates) were extracted using Trizol reagent (Invitgen, Waltham, MA, USA). Then, total RNA was treated using Turbo DNase I (Ambion, Austin, TX, USA) for 30 min and purified using RNeasy® Plant Mini Kit (QIAGEN, Hilden, Germany). Then, the TruSeq RNA sample Prep V2 kit (Illumina, San Diego, CA, USA) was used to construct a transcriptome-sequencing cDNA library and the Agilent 2100 TapeStation (Agilent, Santa Clara, CA, USA) system was used to detect the quality of the cDNA library. Finally, paired-end sequencing was performed on the Illumina NovaSeq 6000 sequencer by Beijing Biomics Biotech Co., Ltd. (Beijing, China). After the sequencing was completed, we uploaded the data to the NCBI database under the BioProject number PRJNA783478.
10
Panda Sperm RNA Extraction and Sequencing
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Prior to total RNA extraction, semen was treated with 0.5% Triton X-100 according to previous study to eliminate somatic cell contamination [25 (link)]. For each panda sample (n = 5), total RNA extraction of fresh and frozen-thawed sperm was performed with Trizol LS Reagent (Invitrogen, Carlsbad, CA). RNA concentration, purity and RNA integrity were measured using the NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA) and the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 System (Agilent Technologies, Santa Clara, CA, USA), respectively. Then, miRNA libraries were constructed using NEBNext Ultra-small RNA Sample Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) according to manufacturer’s instructions. The quality and yield after sample preparation were determined with Agilent 2100 Tape Station and Qubit 2.0, and the RNA was sequenced on an Illumina Hiseq 2500 platform.
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