Male mice, WT and Asgr1–/–, were fasted overnight before collection of the liver tissues. Total RNA of each sample was extracted using TRIzol Reagent/RNeasy Mini Kit (QIAGEN). Total RNA of each sample was quantified and qualified by Agilent 2100/2200 Bioanalyzer (Agilent Technologies), with NanoDrop (Thermo Fisher Scientific). A total of 1 μg RNA was used for the following library preparation. Next-generation sequencing library preparations were constructed according to the manufacturer’s protocol. The sequences were processed and analyzed by GENEWIZ. All the sequencing data have been submitted to the National Center for Biotechnology Information’s Gene Expression Omnibus data bank and the access number is GSE178370.
Trizol reagent rneasy mini kit
Manufactured by Qiagen
Sourced in Germany
TRIzol Reagent/RNeasy Mini Kit is a laboratory product that serves as a tool for the extraction and purification of RNA from biological samples. It enables the isolation of high-quality RNA for use in various downstream applications, such as gene expression analysis, RT-PCR, and Northern blotting. The kit provides a reliable and efficient method for RNA extraction and purification.
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Lab products found in correlation
Nanodrop, by Thermo Fisher Scientific (4 mentions)
Agilent 2100 2200 bioanalyzer, by Agilent Technologies (2 mentions)
Hiseq novaseq instrument, by Illumina (2 mentions)
Qsep100, by Bioptic (1 mentions)
Human cd4 t cell isolation kit, by BD (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
6 protocols using trizol reagent rneasy mini kit
1
Transcriptomic analysis of Asgr1-deficient mice
2
Transcriptome Analysis of Modified Atmosphere Preservation
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The samples from the 1d (bx(MAP)1, ck(control)1), 4d (bx2, ck2), 8d (bx3, ck3) and 14d (bx4, ck4) of the 10 % O2 + 5% CO2 + 85% N2 modified atmosphere preservation experiment at 4 °C were selected, and the low-temperature preservation at 4 °C during the same period was used as the control for transcriptomic analysis. There were three replicate samples per group.
Total RNA of each sample was extracted using TRIzol Reagent/RNeasy Mini Kit (Qiagen)/other kits. Total RNA of each sample was quantified and qualified by Agilent 2100/2200 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc., Waltham, MA, USA). An amount of 1 μg total RNA was used for following library preparation. In order to remove technical sequences, including adapters, polymerase chain reaction (PCR) primers, or fragments thereof, and quality of bases lower than 20, pass filter data of fastq format were processed by Cutadapt (v1.9.1) to become high-quality, clean data. Differential expression analysis used the DESeq2 Bioconductor package (v1.6.3), p-adjusted (Padj) of genes was set <0.05 to detect differentially expressed genes. GOSeq (v1.34.1) was used to identify Gene Ontology (GO) terms. KEGG (Kyoto Encyclopedia of Genes and Genomes) was used to enrich significant differential expression genes in KEGG pathways.
Total RNA of each sample was extracted using TRIzol Reagent/RNeasy Mini Kit (Qiagen)/other kits. Total RNA of each sample was quantified and qualified by Agilent 2100/2200 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA), NanoDrop (Thermo Fisher Scientific Inc., Waltham, MA, USA). An amount of 1 μg total RNA was used for following library preparation. In order to remove technical sequences, including adapters, polymerase chain reaction (PCR) primers, or fragments thereof, and quality of bases lower than 20, pass filter data of fastq format were processed by Cutadapt (v1.9.1) to become high-quality, clean data. Differential expression analysis used the DESeq2 Bioconductor package (v1.6.3), p-adjusted (Padj) of genes was set <0.05 to detect differentially expressed genes. GOSeq (v1.34.1) was used to identify Gene Ontology (GO) terms. KEGG (Kyoto Encyclopedia of Genes and Genomes) was used to enrich significant differential expression genes in KEGG pathways.
3
Bulk RNA-seq Library Preparation
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Total RNA was extracted using the TRIzol reagent/RNeasy minikit (Qiagen). rRNA was depleted from total RNA using the rRNA removal kit. The depleted rRNA was then fragmented and reverse transcribed. The first cDNA strand was synthesized using ProtoScript II reverse transcriptase with random primers and actinomycin D. The second cDNA strand was synthesized using the second-strand synthesis enzyme mix. dA tailing was performed in one reaction, followed by a T-A ligation to add adaptors to both ends. Size selection of adaptor-ligated DNA was then performed using beads, and fragments of ~400 bp were recovered. Each sample was then amplified by PCR using the P5 and P7 primers. The PCR products were validated using a Qsep100 (Bioptic, Taiwan, China) and quantified using the Qubit 3.0 fluorometer. Libraries with different indices were multiplexed and loaded on an Illumina HiSeq/NovaSeq instrument according to the manufacturer’s instructions. Sequencing was carried out using a 2 × 150-bp paired-end-read configuration.
4
Transcriptomic Analysis of S. avermitilis
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Sample RNA extraction was performed using the TRIzol Reagent /RNeasy Mini Kit (Qiagen) kit. cDNA synthesis was performed according to the kit SPARK script II RT Plus Kit (With gDNA Eraser). qRT-PCR analysis was used to determine the transcript levels of various genes [38 (link)]. Transcription of housekeeping gene 16S rRNA was used as internal control. Each experiment was performed in triplicate. S. avermitilis MA-4680 (BA000030.4) as a reference genome.
5
Profiling Cochlear Transcriptome by RNA-Seq
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Total RNA was extracted from the cochleae using TRIzol Reagent/RNeasy Mini Kit (Qiagen, Hilden, Germany) and quantified with an Agilent NanoDrop (ThermoFisher Scientific). A total of 1 μg RNA from each sample was used for the library preparations for next generation sequencing, and these were constructed according to the manufacturer’s protocol. The cDNA was synthesized by ProtoScript II Reverse Transcriptase and Strand Synthesis Enzyme Mix, and then treated with End Prep Enzyme Mix to repair both ends and to add tail-A, which followed by a ligation to add adaptors. Each sample was then amplified by PCR using P5 and P7 primers, with both primers carrying sequences that can anneal with the flow cell to perform bridge PCR and carrying indexes that allow for multiplexing. The libraries were then multiplexed and loaded onto an Illumina HiSeq/Novaseq instrument according to the manufacturer’s instructions (Illumina, San Diego, CA). The expression levels of all mapped genes were estimated by stringtie (v2.0), and the gene expression levels were determined by fragments per kilobase of transcript per million fragments mapped. Differential expression analysis was based on the DESeq2 Bioconductor package, and p values of less than 0.05 indicated DEGs. The analysis of pathways was based on the Kyoto Encyclopedia of Genes and Genomes database.
6
Isolation and Purification of CD4+ T Cells from Human PBMCs
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Peripheral blood mononuclear cell (PBMCs) were isolated from HCs and SLE patients using human peripheral blood lymphocyte separation medium (Tianjin HaoYang Biological Manufacture, Tianjin, China) within 6 h of sample collection. CD4+ T cells were then purified following immunomagnetic separation using a human CD4+ T cell isolation kit (BD Biosciences, San Jose, CA, United States) according to the manufacturer’s instructions. For RNA purification, the isolated human CD4+ T cells were lysed in TRIzol reagent (Invitrogen Life Technologies, Grand Island, NY, United States). The extracted RNA was further digested by DNase I (Invitrogen, Waltham, MA, United States) to remove residual DNA, and subsequently separated from each sample using TRIzol reagent/RNeasy Mini kit (Qiagen, Hilden, Germany). Total extracted RNA was stored for use at −80°C.
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