Total RNA was extracted using RNeasy Plus Micro Kit (Qiagen #74034) according to the manufacturer’s protocol. RNA quality was checked on Bioanalyzer (Agilent) using RNA 6000 Nano Kit (Agilent #5067-1511). Total RNA samples with RIN value > 7 were used for library preparation. cDNA libraries were prepared using TruSeq RNA Sample Prep v2 (Illumina #RS-122-2001) and subsequent next-generation sequencing was performed on an Illumina HiSeq 4000 platform by Macrogen INC. Sequencing from each sample was performed: 101nt long pair-end reads with at least 80 M reads per sample. Differential expression analysis was performed on the Galaxy platform by the following pipeline: Paired-End sequences obtained from the HiSeq 4000 platform were trimmed using the Trimmomatic v0.36.3 [61 (link)]. Sequence reads that pass quality filters were mapped to the human reference genome hg38 using Bowtie 2 v2.3.4.1 [62 (link)]. Mapped reads were counted using featureCounts v1.6.0.3 [63 (link)] followed by calculating differential gene expression with DESeq2 v2.11.40.1 [64 (link)]. Differentially expressed transcripts were filtered according to the parameters of log2FC ≥ 0.5 for upregulated and log2FC ≤ −0.5 for downregulated genes. Adjusted p-value ≤ 0.01 was considered as significant.
Truseq rna sample prep v2
Manufactured by Illumina
Sourced in United States
The TruSeq RNA Sample Prep v2 is a library preparation kit designed for transcriptome sequencing. It is used to prepare high-quality RNA samples for next-generation sequencing on Illumina platforms. The kit includes reagents and protocols for converting RNA into cDNA libraries that are ready for sequencing.
Automatically generated - may contain errors
Lab products found in correlation
Bioanalyzer, by Agilent Technologies (3 mentions)
Rna 6000 nano kit, by Agilent Technologies (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Hiseq 2500 sequencer, by Illumina (3 mentions)
Rneasy plus micro kit, by Qiagen (2 mentions)
Hiseq 4000 platform, by Illumina (2 mentions)
Gaiix, by Illumina (2 mentions)
Hiseq 2000 instrument, by Illumina (1 mentions)
Encore rapid library system, by Tecan (1 mentions)
Ovation rna seq system v2, by Tecan (1 mentions)
Bioanalyzer dna 1000 kit, by Agilent Technologies (1 mentions)
Hiseq 2000 sequencing platform, by Illumina (1 mentions)
Rneasy spin column, by Qiagen (1 mentions)
Quant it picogreen assay, by Thermo Fisher Scientific (1 mentions)
Hiscansq sequencer, by Illumina (1 mentions)
Humanmethylation450 beadchip, by Illumina (1 mentions)
Rneasy mini kit, by Qiagen (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Agilent bioanalyzer, by Agilent Technologies (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
15 protocols using truseq rna sample prep v2
1
Transcriptome Profiling Using Illumina HiSeq
2
RNA-seq Library Preparation for Tissues
3
Midgut Transcriptome Library Preparation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Individual midgut libraries were prepared from total RNA extracts from individual midguts after quality control with a Bioanalyzer RNA 6000 kit (Agilent). Purification and fragmentation of mRNA, cDNA synthesis, end-repair, A-tailing, Illumina indexes ligation and PCR amplification were performed using TruSeq RNA Sample Prep v2 (Illumina) followed by cDNA quality check by Bioanalyzer DNA 1000 kit (Agilent). Libraries were diluted to 10 pM after Qubit quantification (ThermoFisher), loaded onto a flow cell, clustered with cBOT (Illumina). Single-end reads of 51 nucleotides in length were generated on a HiSeq2000 sequencing platform (Illumina). Sequencing reads with a quality score <30 were trimmed using Cutadapt [61 ]. Passing-filter reads were mapped to Ae. aegypti transcripts (AaegL3.1, http://vectorbase.org ) using Bowtie2 [59 (link)] with the “sensitive” option. They were processed with the Samtools suite [62 (link)] to create of a matrix of raw counts used for gene expression analysis. The RNA-Seq data were deposited to SRA under accession number PRJNA386455 (https://www.ncbi.nlm.nih.gov/bioproject/386455 ).
4
RNA Sequencing of Tumor Tissues
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA was isolated from fresh frozen tumor tissues using the RNeasy spin-column method (Qiagen, Milan, Italy). RNA libraries were prepared from 500 ng total RNA in accordance with Illumina's TruSeq RNA Sample Prep v2 protocol (Illumina, San Diego, California). Libraries were quality checked and sized with Agilent DNA 7500 chips on the Bioanalyzer 2100 (Agilent Technologies, Taiwan), then quantified using Quant-IT picogreen assay (Life Technologies). Paired-end libraries were sequenced at 2 × 80 bp read length, on the HiScanSQ Illumina sequencer. An average of 80.5 million reads per sample were analyzed for each sample, corresponding to 6.5 Gb of sequences/sample.
5
Comprehensive Genomic Analysis of B-ALL
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Samples S1–S5 and A1d–A1r, A2d–A2r, and A3d–A3r underwent DNA methylation analysis using the HumanMethylation450 BeadChip (Illumina, San Diego, CA, USA). The total RNA of four B-ALL patients at diagnosis (A1d, A2d, A3d, and A4d) and the same four patients at remission (A1r, A2r, A3r, and A4r) were also processed using the TruSeq RNA Sample Prep V2 (Illumina, San Diego, CA, USA) to perform whole-transcriptome analysis. Additional information and statistical analysis are presented in Appendix A.1 .
6
RNA-seq of Pooled Samples across Strains
7
RNA-seq and ChIP-seq of Islet Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total RNA extraction from purified islet is described above. mRNA from whole islets was used to generate libraries using Illumina TruSeq RNA Sample Prep v2 (RS-122-2001). The manufacturer’s recommendations were followed and the libraries were sequenced on an Illumina HiSeq 2500 sequencer. All sequence data were performed in biological triplicates, each containing islets from 1-2 animals, at 2 x 50 bp length with high quality metrics (>20 Phred score) and nucleotide distribution. For 25 weeks βEedKO samples, two biological replicates were used, each containing islets from at least 4 animals.
NEXSON ChIP-seq workflow (Arrigoni et al., 2015 (link)) is applied to freshly isolated mouse islets. ChIP-seq performed using antibodies against H3K27me3 (Diagenode, #C15410195), H3K9me3 (Diagenode, #C15410193), H3K27ac (Diagenode, #C15410196), H3K4me3 (Diagenode, pAb-003-050), H2AK119ub (Cell signaling, #8240) and Pol-II (Diagenode, #C1520004). A list of ChIP-seq data sets used can be found in theKey Resources Table and Table S5 .
NEXSON ChIP-seq workflow (Arrigoni et al., 2015 (link)) is applied to freshly isolated mouse islets. ChIP-seq performed using antibodies against H3K27me3 (Diagenode, #C15410195), H3K9me3 (Diagenode, #C15410193), H3K27ac (Diagenode, #C15410196), H3K4me3 (Diagenode, pAb-003-050), H2AK119ub (Cell signaling, #8240) and Pol-II (Diagenode, #C1520004). A list of ChIP-seq data sets used can be found in the
8
RNA-seq Analysis of Differentially Expressed Genes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA was extracted using the RNeasy Mini Kit (QIAGEN) on the six samples at the same time. Extracted RNA samples underwent quality control assessment using Bioanalyzer (Agilent, Santa Clara, CA, USA) and were quantified using NanoDrop from NanoDrop Technologies (Wilmington, DE, USA). RNA libraries were prepared using the Illumina TruSeq RNA sample prep V2 with ribosomal RNA depletion and were sequenced using HiSeq 2500 sequencer (Illumina Inc., San Diego, CA, USA) at the Center for Applied Genomics at the Children’s Hospital of Philadelphia per standard protocols (paired-end 100 base pairs). The RNA-seq data were aligned on the hg19 reference genome using STAR (www.encodeproject.org/software/star/ ) and processed using Cufflinks (http://cole-trapnell-lab.github.io/cufflinks/ ). For each gene, we compared the expression levels between cases and controls. A gene was considered differentially expressed if the P value is less than 0.05 and fragments per kilobase of transcript per million mapped reads has at least twofold change. To identify overrepresented functional categories among genes that are differentially expressed, we performed annotation analysis using the David Functional Annotation Resource (https://david.ncifcrf.gov/ ). Multiple testing was adjusted using the Benjamini-Hochberg approach, and enrichment was declared if the adjusted P value is less than 0.05.
9
RNA Isolation and Sequencing From Liver
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
RNA from liver of control (ERα floxed) and LERKO mice was isolated with TRIzol (Invitrogen) and purified using the RNeasy minikit protocol (Qiagen), according to the manufacturer’s instructions.
RNA Quality Control was performed with the RNA 6000 Nano Kit (Agilent) on Agilent Bioanalyzer (Agilent). The RNA Integrity Number (RIN) was determined for every sample and all samples were considered suitable for processing if RIN > 7.5. RNA concentration was spectrophotometrically estimated using Nanoquant Infinite M200 instrument (Tecan). Sequencing libraries were prepared using the TruSeq RNA Sample Prep V2 (Illumina) with an input of 800 ng of total RNA. We validated and quantified final libraries with the DNA1000 kit on Agilent Bioanalyzer. Pooled libraries were sequenced on Illumina NextSeq, producing 2X75 bp paired end reads.
RNA Quality Control was performed with the RNA 6000 Nano Kit (Agilent) on Agilent Bioanalyzer (Agilent). The RNA Integrity Number (RIN) was determined for every sample and all samples were considered suitable for processing if RIN > 7.5. RNA concentration was spectrophotometrically estimated using Nanoquant Infinite M200 instrument (Tecan). Sequencing libraries were prepared using the TruSeq RNA Sample Prep V2 (Illumina) with an input of 800 ng of total RNA. We validated and quantified final libraries with the DNA1000 kit on Agilent Bioanalyzer. Pooled libraries were sequenced on Illumina NextSeq, producing 2X75 bp paired end reads.
10
Transcriptomic Analysis of Anxiety-like Behavior in Zebrafish
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
We further carried out a transcriptomic analysis of brain samples from a publicly available dataset (accession number GSE61108) of adult zebrafish with different inherent levels of anxiety-like behavior. The strains in the dataset included AB (also used in the above experiments), which is a low anxiety-like strain, common in laboratories, and two other strains with higher anxiety-like behavior, termed here collectively as SB, comprising LSB (low stationary behavior) and HSB (high stationary behavior) [71 (link),72 (link)]. Each strain was represented by four pooled samples of ten whole brains (two males and two females); thus, for our analysis, there were four AB samples and eight SB samples (four HSB and four LSB). cDNA libraries had been generated with TruSeq RNA Sample Prep V2 of Illumina and sequenced as 72 bp single ends on Illumina (GAIIX), similar to our procedures. To analyze this dataset, we used a similar procedure to the RNA analysis for larval samples, with one exception. FastQC, version v0.11.9 revealed TruSeq adapters, which we removed with Trimmomatic v.0.36 and further confirmed removal by FastQC analysis. The remaining analysis was executed with RSEM and DESeq2, as described above.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!