Total RNA was extracted from individual samples by Direct-zol RNA MiniPrep Plus kit (Zymoresearch, Irvine, CA, USA) according to manufacturer’s protocol. Concentration of DNA-digested RNAs was measured by NanoDrop™ One Microvolume UV-Vis Spectrophotometer (Thermo Scientific, Waltham, MA, USA). The cDNA library preparation for RNASeq and small RNASeq, and sequencing on illumina HiSeq 4000 was performed by GENEWIZ (South Plainfield, NJ, USA).
Hiseq 4000
Manufactured by Genewiz
Sourced in United States
The HiSeq 4000 is a high-throughput sequencing system designed for large-scale genomic research. It features advanced optics and fluidics technologies to deliver rapid and accurate DNA sequencing results. The HiSeq 4000 is capable of generating up to 1.5 terabases of data per run, making it a powerful tool for a variety of genomic applications.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (4 mentions)
Rnalater, by Thermo Fisher Scientific (4 mentions)
Qubit rna assay, by Thermo Fisher Scientific (2 mentions)
Rneasy kit protocol, by Qiagen (2 mentions)
Rna pure link mini kit, by Thermo Fisher Scientific (2 mentions)
Casava, by Illumina (2 mentions)
Precellys, by Bertin Technologies (2 mentions)
Bioanalyzer, by Agilent Technologies (2 mentions)
Rna 6000 nano chip, by Agilent Technologies (2 mentions)
Nanodrop one microvolume uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Direct zol rna miniprep plus kit, by Zymo Research (1 mentions)
Kk8481, by Illumina (1 mentions)
5 protocols using hiseq 4000
1
RNA Extraction and Sequencing
2
Testes RNA Extraction and Sequencing
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Testes were harvested and homogenized in Trizol reagent (Thermo Fisher) and stored at −80°C prior to processing. Total RNA was extracted from the aqueous phase, mixed with ethanol and purified using the RNeasy kit protocols and reagents (Qiagen). RNA was quantified using the Qubit RNA assay (Thermo Fisher) and 400–500 ng total RNA per sample was used in stranded mRNA-seq library preparation (KAPA Biosystems, KK8481) for Illumina sequencing. Libraries were pooled and sequenced with 2×150 cycles paired-end to an average depth of 19.4 million reads per sample on a HiSeq 4000 by Genewiz (South Plainfield, NJ, USA).
3
Transcriptional Profiling of Meningeal Tissue
4
Transcriptional Profiling of Meningeal Tissue
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Meningeal tissue was stabilized in RNAlater (Thermo Fisher) and then placed into lysis buffer and homogenized using a Precellys (Bertin instruments). RNA was subsequently extracted using the RNA pure link mini kit (Thermo Fisher) as per manufacturer’s instructions. RNA integrity was assessed using an RNA 6000 nano chip on a Bioanalyzer (Agilent). Libraries were prepared as per manufactures instructions using the TruSeq Stranded Total RNA kit with RiboZero. Libraries were checked for size with a high sensitivity DNA chip on a Bioanalyzer (Agilent), quantified using Kappa library quantification kit (Kappa biosystems) and pooled at an equimolar ratio. Library pools were sequenced on a Hiseq 4000 by Genewiz.
Data were demultiplexed using Casava (Illumina) to generate Fastq files. These were aligned to the mm10 genome with Hisat2 and a counts table produced using Featurecounts (RSubread). Normalization and differential expression analysis were carried out using DESeq2 and pathways analysis using the GSEA java application (Broad) against pathways found within the MSigDB database.
Sequencing data has been made available on GEO under accession number GSE135620 and GSE135733.
Data were demultiplexed using Casava (Illumina) to generate Fastq files. These were aligned to the mm10 genome with Hisat2 and a counts table produced using Featurecounts (RSubread). Normalization and differential expression analysis were carried out using DESeq2 and pathways analysis using the GSEA java application (Broad) against pathways found within the MSigDB database.
Sequencing data has been made available on GEO under accession number GSE135620 and GSE135733.
5
Transcriptome Profiling of Mouse Testes
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P1 or P3 testes were harvested and homogenized in Trizol reagent (Thermo Fisher) and stored at -80°C prior to processing. Total RNA was extracted from the aqueous phase, mixed with ethanol and purified using the RNeasy kit protocols and reagents (Qiagen). RNA was quantified using the Qubit RNA assay (Thermo Fisher) and 400–500 ng total RNA per sample was used in stranded mRNA-seq library preparation (KAPA Biosystems, KK8481) for Illumina sequencing. Libraries were pooled and sequenced with 2x150 cycles paired-end to an average depth of 26 million reads per sample on a HiSeq 4000 by Genewiz (South Plainfield, NJ). Reads were trimmed using Trim Galore (v0.6.0) and cutadapt (v1.18) and assessed for quality with FastQC (v0.11.8). Trimmed reads were mapped with STAR (v2.7.2a) to the GRCm38 (mm10) genome. The GENCODE M25 gene annotation set was used to estimate strand-specific gene expression data with the Bioconductor package RSubread (v2.2.6). Differentially expressed genes were identified using DESeq2 (v1.28.1).
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