Solexa libraries were prepared using the NEBNext DNA Sample Preparation Kit (NEB) and Illumina PE adapters (Illumina) from three pooled replicates of control cell line and TRβ-expressing cells +/−T3. Libraries were sequenced on a Solexa GAIIx following standard procedures. Solexa sequencing data was aligned to HG19 with Bowtie 0.12.7 [46] (link). Binding peaks were analyzed using QuEST 2.4 [47] (link).
Pe adapters
Manufactured by Illumina
PE adapters are laboratory equipment used to prepare DNA samples for sequencing on Illumina platforms. They facilitate the ligation of sequencing adapters to DNA fragments, which is a crucial step in the library preparation process. PE adapters are designed to be compatible with Illumina's paired-end sequencing technology, enabling the generation of sequence data from both ends of the DNA fragments.
Automatically generated - may contain errors
Lab products found in correlation
Cbot cluster generation system, by Illumina (3 mentions)
Hiseq 2000 platform, by Illumina (2 mentions)
End it dna end repair kit, by Illumina (2 mentions)
Superscript double stranded cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Hiseq platform, by Illumina (2 mentions)
Dynabeads mrna purification kit, by Thermo Fisher Scientific (2 mentions)
Gaiix, by Illumina (2 mentions)
Nebnext dna sample preparation kit, by New England Biolabs (1 mentions)
Uracil dna glycosylase, by New England Biolabs (1 mentions)
Dynabeads oligo dt 25, by Thermo Fisher Scientific (1 mentions)
Nebnext rna first strand synthesis module, by New England Biolabs (1 mentions)
Nebnext ultra directional rna second strand synthesis module, by New England Biolabs (1 mentions)
Agencourt rnaclean xp beads, by Beckman Coulter (1 mentions)
Hiseq 2000, by Illumina (1 mentions)
Phusion high fidelity pcr master mix, by New England Biolabs (1 mentions)
Paired end protocol, by Illumina (1 mentions)
Cluster station, by Illumina (1 mentions)
Hiseq 2000 sequencing platform, by Illumina (1 mentions)
Platinum taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Nextflex bisulfite library prep kit for sequencing, by Illumina (1 mentions)
10 protocols using pe adapters
1
ChIP-seq protocol for TRβ-expressing cells
2
Directional RNA-seq Library Preparation
3
Whole-Transcriptome RNA Sequencing for Gene Discovery
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Concurrently with the above study, patients at the Texas Children's Cancer Center are being enrolled in a second IRB-approved protocol for prospective clinical whole exome sequencing. A sub-study of this protocol includes permission to perform other genomic and RNA analyses for gene discovery. Whole-transcriptome RNA sequencing (RNA-seq) was performed using high quality total RNA (RIN>7) extracted from fresh-frozen tissue to prepare strand-specific, poly-A+ RNA-seq libraries for sequencing on the Illumina platform (Illumina Inc., San Diego, CA). Briefly, poly-A+ mRNA was extracted from 1 μg total RNA, followed by fragmentation and first strand cDNA synthesis. The resultant cDNA was end-repaired, A-tailed and ligated with Illumina PE adapters. The libraries were sequenced in paired-end mode (2 × 100-bp reads) on an Illumina HiSeq 2000 platform following amplification on the cBot cluster generation system (Illumina Inc.). On average, over 84 million paired-end reads were generated per sample.
4
Whole-Transcriptome RNA Sequencing for Gene Discovery
5
Illumina-Based Paired-End Sequencing Protocol
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Libraries were constructed using a custom paired-end protocol (Illumina) (Falconer et al. 2012 (link)). Briefly, samples were end-repaired and A-tailed, and Illumina PE adapters were ligated. Libraries were amplified using indexed PE primers for eight to 10 PCR cycles. Amplified indexed libraries were pooled (four to six libraries per pool) and size-selected for paired-end sequencing. A detailed library construction procedure is presented in the Supplemental Material. Cluster generation and paired-end sequencing (100-base-pair [bp] reads) were performed on the Illumina cluster station and Illumina HiSeq 2000 sequencing platform. Sequence reads were mapped to mm9 (NCBI 37) using BWA (Li and Durbin 2009 (link)). Reads passing Illumina’s default chastity filter (Li et al. 2009 (link)) were used to generate library statistics.
6
RNA-Seq Data Quality Control and Processing
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The quality control and reference mapping of the RNA-Seq data were carried out using open-source tools in Ubuntu command-line interface. First, the quality of the raw reads was evaluated using FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/ ) commands. Then, the raw reads were processed using ‘trimmomatic’ package for trimming the Illumina PE adapters (TruSeq3-PE-2.fa:2:30:10), removing the low-quality or poly-N bases from the ends that were below quality score 3, removing the sequences when the average quality per base dropped below 15 in a 4-base sliding window, and dropping the reads with less than 25 bases in length. This led to a set of clean and high-quality RNA-seq reads for subsequent analyses.
7
Quantifying mRNA Expression from RNA-Seq Data
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RNA was harvested from ES cells and EBs as described above. RNA-Seq was performed as previously described [20 (link), 41 (link)]. RNA was harvested from ES cells and EBs as described above. mRNA was purified using a Dynabeads mRNA purification kit (Invitrogen). Double-stranded cDNA was generated using a SuperScript Double-Stranded cDNA synthesis kit (Invitrogen). cDNA was end-repaired using the End-It DNA End-Repair kit (Epicentre), followed by addition of a single A nucleotide, and ligation of PE adapters (Illumina) or custom-indexed adapters. PCR was performed using Phusion High-Fidelity PCR master mix. RNA-Seq libraries were sequenced on Illumina GAIIX or HiSeq platforms according to the manufacture’s protocol.
The RPKM measure (reads per kilo bases of exon model per million reads) proposed previously [62 (link)] was used to quantify the mRNA expression level of a gene from RNA-Seq datasets. Differentially expressed genes were identified using EdgeR (FDR < 0.001 and FC > 2) [63 (link)]. Genes with RPKM < 3 in both conditions in comparison were excluded from this analysis.
The RPKM measure (reads per kilo bases of exon model per million reads) proposed previously [62 (link)] was used to quantify the mRNA expression level of a gene from RNA-Seq datasets. Differentially expressed genes were identified using EdgeR (FDR < 0.001 and FC > 2) [63 (link)]. Genes with RPKM < 3 in both conditions in comparison were excluded from this analysis.
8
Transcriptome Analysis of Human Samples
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All fastq files were trimmed using Trimmomatic (https://github.com/usadellab/Trimmomatic ) using the Illumina PE adapters. The trimmed reads were assessed with FastQC (https://github.com/s-andrews/FastQC ) and then passed through the following analytical pipeline: transcript pseudoalignment and quantification was performed with Salmon (https://github.com/COMBINE-lab/salmon ) using an index generated from the GENCODE version 32 transcriptome using standard arguments, trimmed reads were aligned to a Homo sapiens genome assembly GRCh38 (hg38) using STAR (https://github.com/alexdobin/STAR ) with default arguments using a previously described 2-pass approach. Salmon output was imported into a DESeq object using tximport and differential expression analysis was performed with DESeq2 using standard arguments. Differentially expressed genes were called with FDR-corrected p (p-adj) ≤0.01 and fold change >2 cutoffs, and results were visualized in R.
9
Bisulfite Sequencing Library Preparation
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Following DNA extraction, Bioo Scientific NEXTflex Bisulfite Library Prep Kit for Illumina Sequencing (PerkinElmer, Austin, TX) was used for library preparation. To maximize coverage, we employed two separate restriction digests with MspI and TaqαI. Following digestion, products were pooled, and Klenow Fragment was utilized to create 3’A overhangs. DNA was subsequently purified with Zymo DNA Clean and Concentrate Columns (Irvine, CA) followed by ligation of Methylated Illumina PE Adapters and Ampure purification with SPRI beads. Purified products were Sodium Bisulfite Converted using ZymoResearch EZ DNA Methylation Gold Kit, and libraries were amplified over 20 cycles using Platinum Taq DNA polymerase (ThermoFisher, Waltham, MA), followed by a final Ampure purification (Beckman Coulter, Indianapolis, IN) and confirmation of library size range on a 2% agarose gel. DNA was submitted to the Huntsman Cancer Institute High Throughput genomic core for sequencing on a Hi-Seq 2500 (Illumina, San Diego, CA) using 50 cycle-single read chemistry. Four to six samples were sequenced per lane for a minimum of 35-million reads per sample.
10
RNA-Seq Analysis of shSmyd5 Cancer Cells
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