To evaluate the impact of MAPT p.R406W on lysosomal genes, we queried summary statistics from our previously generated bulk RNAseq data of the insular cortex of MAPT p.R406W (n = 2) and neuropathology-free controls (n = 2) [19 (link)]. Briefly, DNA libraries of individual samples were constructed using the TruSeq Stranded Total RNA Sample Prep with Ribo-Zero Gold kit (Illumina) and then sequenced by the HiSeq 4000 (Illumina). FASTQ files were aligned to human GRCh37 primary assembly. After alignment, Salmon (v. 0.7.2) was used to quantify expression levels of individual genes included in the GENCODE reference genome (GRCh37.75). Differential gene expression was performed using the R (v.3.4.2) package DESeq2 (v.1.18.1) as previously described [19 (link)]. From the summary statistics, we extracted genes that are regulated by Transcription Factor EB (TFEB), defined as: [1 (link)] containing a Coordinated Lysosomal Expression and Regulation (CLEAR) sequence or [2 (link)] being altered by overexpression of TFEB in vitro [37 (link)]. To determine whether genes that are regulated by TFEB and altered in MAPT p.R406W brains were enriched in specific functional pathways, gene enrichment analysis was performed using ToppGene [38 (link)].
Truseq stranded total rna sample prep kit with ribo zero gold
Manufactured by Illumina
The TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero Gold is a laboratory equipment product designed for total RNA sample preparation. It enables the removal of ribosomal RNA (rRNA) from total RNA samples, allowing for the enrichment of other RNA species, such as mRNA, for subsequent analysis.
Automatically generated - may contain errors
Lab products found in correlation
Rneasy mini kit, by Qiagen (3 mentions)
Hiseq 4000, by Illumina (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Nanodrop spectrophotometer, by Thermo Fisher Scientific (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Hiseq 2500, by Illumina (2 mentions)
Tissuelyser lt, by Qiagen (1 mentions)
Quant it rna assay, by Thermo Fisher Scientific (1 mentions)
Qubit fluorometer, by Thermo Fisher Scientific (1 mentions)
Allprep dna extraction kit, by Qiagen (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Truseq small rna sample prep kit, by Illumina (1 mentions)
Qiazol, by Qiagen (1 mentions)
Mirneasy mini kit, by Qiagen (1 mentions)
8 protocols using truseq stranded total rna sample prep kit with ribo zero gold
1
Investigating MAPT p.R406W Impact on Lysosomal Genes
2
RNA Extraction and Sequencing of Brain Tissues
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RNA was extracted from frozen brain tissues and iPSC-derived neuronal cell pellets using Tissue Lyser LT and RNeasy Mini Kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. RNA yields of individual samples were quantified by the Quant-iT RNA Assay (Life Technologies) on the Qubit Fluorometer (Fisher Scientific). Quality of yielded RNA of individual samples was assessed by the DV200 metric. DV200 of individual samples was measured with the RNA 6000 Pico Assay using the Bioanalyzer 2100 (Agilent Technologies). cDNA libraries of individual samples were constructed using the TruSeq Stranded Total RNA Sample Prep with Ribo-Zero Gold kit (Illumina) and then sequenced by the HiSeq 4000 (Illumina) at the McDonnell Genome Institute at Washington University in St. Louis with a mean of 58.14 ± 8.62 million reads as previously described23 (link).
3
RNA-seq Library Preparation from Cells
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Total RNA was isolated from ~700K cells utilizing the AllPrep DNA extraction kit (Qiagen). ERCC RNA Spike-In Mix 1 was added to 100–250 ng of total RNA as outlined by the manufacturer (Ambion, Life Technologies). The ERCC control mix is a set of external RNA controls that enable performance assessment for gene expression experiments. The cDNA library was prepared with the TruSeq Stranded Total RNA Sample Prep with Ribo-Zero Gold kit (Illumina). The concentration of each cDNA library was determined through qPCR (Kapa Biosystems). In all, 2 × 150 reads were generated on the HiSeq4000/NovaSeq6000 instrument (Illumina) generating ~83 million read pairs/sample.
4
RNA Sequencing of Muscle Samples
5
RNA-seq Analysis of Preterm Birth
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We used publicly available RNA sequencing data (GEO dataset ID: GSE73714)33 (link) from paired villous trophoblast and decidua basalis specimens collected from spontaneous idiopathic preterm birth (SPTB; gestation age 30–33 weeks, n = 5) or term birth (by caesarean section, absence of labor; i.e., ETB; gestation age 38–39 weeks, n = 5). Details of the RNA sequencing and data generation were described previously33 (link). In short, total RNA was extracted from flash-frozen specimens with TRIzol. RNA-seq libraries were constructed with the TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero Gold (Illumina) and sequenced with the Illumina HiSeq 2500 platform. Illumina Analysis pipeline in HiSeq Control Software v2.2.38 was used for image analysis, base calling, and error estimation. Reads were mapped to UCSC hg38, and data were normalized by using Bioconductor statistical packages (https://www.bioconductor.org/ ).
6
Muscle RNA Sequencing Protocol
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Before using the muscle samples for RNA sequencing, C.I. and J.S. inspected the hematoxylin and eosin‐stained sections of the muscle samples and confirmed that there were < 1% fibers with ice artifact within each section. Ten frozen muscle slices with 50 μm thickness, which were kept at −80°C, were homogenized in 0.75 ml of Trizol at 4°C. RNA was isolated using RNeasy Mini Kit (Qiagen, Venlo, the Netherlands). Concentrations of RNA were measured by Nanodrop spectrophotometer (Thermo Fisher Scientific, Waltham, MA). The integrity of RNA was analyzed by Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA). The RNA integrity numbers of these samples were confirmed to be > 6, and the interquartile ranges were from 7.6 to 8.2.
Sequence libraries were prepared after removal of ribosomal RNA using TruSeq Stranded Total RNA Sample Prep kit with Ribo‐Zero Gold (Illumina, San Diego, CA).
Sequence libraries were prepared after removal of ribosomal RNA using TruSeq Stranded Total RNA Sample Prep kit with Ribo‐Zero Gold (Illumina, San Diego, CA).
7
Comprehensive RNA Sequencing of USSC
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For sequencing, USSC were lysed in culture dish by adding 750 µl QIAzol (Qiagen, Hilden, Germany) after washing with PBS. RNA from native and XXL-USSC was isolated by miRNeasy Mini Kit (Qiagen). Quality and quantity of RNA samples were measured on a 2100 Bioanalyzer (Agilent, Amstelveen, the Netherlands). Integrity numbers ranged from 8.2 to 10. For RNA sequencing, a maximum of 1 µg total RNA was used for library preparation with TruSeq Small RNA Sample Prep Kit (#RS-200-0012/0024, Illumina, San Diego, CA). 300 ng of total RNA were used for library preparation with TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero Gold (#RS-122-2301, Illumina). Size and DNA concentration of prepared cDNA libraries were also analysed on a 2100 Bioanalyzer. A concentration of seven picomolar of template DNA was loaded per flow cell and high-throughput sequencing was performed on the Illumina HiSeq. 2500 with 50 cycles (miRNAs) or 100 cycles (mRNAs).
8
RNA-Seq Library Preparation and Sequencing
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RNA-seq libraries were prepared using the Illumina TruSeq Stranded Total RNA Sample Prep Kit with Ribo-Zero Gold (catalog #RS-122-2201) according to the manufacturer’s protocol. The sequencing was performed in paired end manner, generating 2X 100 bp paired-end reads using the Illumina HiSeq 2500 system. Pre-alignment data QC were assessed with FastQC. Post-alignment data quality was assessed with an in-house quality control pipeline/database for RNA-seq data51 (link). RNA-seq data were trimmed for any adapter sequences using AdapterRemoval52 (link).
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