HuH-7 cells were transfected with control, FOXA1, or CEBPA siRNAs (10 nM) for 48 h. Total RNA was extracted from the cells using the RNeasy Plus Mini kit. Agilent Bioanalyzer device (Agilent Technologies) was used to assess the quality of extracted RNA. Subsequently, RNA-seq libraries were prepared with total RNA (100 ng) using an NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs) according to the manufacturer’s protocol. The libraries were sequenced with 100 bp paired-end reads on the DNBSEQ-G400RS (MGI Tech, Shenzhen, China). The analysis of sequencing data was performed by a standard RNA-seq analytical pipeline. Briefly, STAR(v2.7.3a)45 (link) was used to align the sequencing data to the human genome (hg38). Quantification of gene expression was performed using RSEM (v1.3.3)46 (link). The DESeq2 package (v1.26.0)47 (link) was used to normalize the read count data and test for differential gene expression.
Agilent bioanalyzer device
Manufactured by Agilent Technologies
Sourced in United States
The Agilent Bioanalyzer device is a microfluidics-based platform used for the automated analysis of biomolecules, such as DNA, RNA, and proteins. It provides rapid, sensitive, and reproducible results through the integration of sample preparation, separation, detection, and data analysis in a single system.
Automatically generated - may contain errors
Lab products found in correlation
Nebnext ultra 2 directional rna library prep kit, by New England Biolabs (2 mentions)
Ribopure rna isolation kit, by Thermo Fisher Scientific (2 mentions)
Superscript 2, by Thermo Fisher Scientific (2 mentions)
Nanodrop equipment, by Thermo Fisher Scientific (2 mentions)
Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions)
Dnbseq g400rs, by MGI Tech (1 mentions)
Tri reagent, by Thermo Fisher Scientific (1 mentions)
Hiseq 2500 platform, by Illumina (1 mentions)
Rneasy plus mini kit, by Qiagen (1 mentions)
4 protocols using agilent bioanalyzer device
1
RNA-seq Analysis of HuH-7 Cells
2
Adipose Tissue RNA Extraction and cDNA Synthesis
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The entire available adipose tissue sample (~50 to 100 mg) from each biopsy was used for total RNA extraction using the RiboPureTM RNA isolation kit (Ambion, Austin, TX, USA), following the manufacturer’s recommendations. The obtained RNA was quantified using a NanoDrop equipment (NanoDrop Technologies, Wilmington, DE, USA), and the RNA quality was assessed with an Agilent bioanalyzer device (Agilent Technologies, Palo Alto, CA, USA). The RNA integrity number values obtained for all the samples were in the range from 7.5 to 8.5. First-strand cDNA synthesis was carried out with Superscript II (Invitrogen Life Technologies, Paisley, UK) and random hexamers in a total volume of 20 μL containing 1 μg of total RNA, following the supplier’s instructions.
3
Placental Transcriptome Analysis of Fetal Growth
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Total RNA was extracted from 50–100 mg of placental chorion obtained from 40 fetuses corresponding to both fetal genders and treatments (10 HTX-males, 10 HTX-females, 10 C-males and 10 C-females). RNA was extracted using RiboPureTM RNA isolation kit (Ambion, Austin, TX, USA) following the manufacturer’s recommendations. Samples were homogenized with 1 mL of TRI Reagent (Ambion, Austin, TX, USA), using an Ultra-Turrax® homogenizer and concentration and quality of the RNA obtained was determined using a NanoDrop equipment (NanoDrop Technologies, Wilmington, DE, USA) and an Agilent bioanalyzer device (Agilent Technologies, Palo Alto, CA, USA). First-strand cDNA synthesis was carried out with Superscript II (Invitrogen Life Technologies, Paisley, UK).
The qPCR assessment included genes involved in antioxidant homeostasis, vascularization and prenatal growth (SOD1, CAT, HIF1A, VEGFA, NOS2, IGF1 and UCP2) and it was performed as previously described for our group [50 (link)]. Stability of endogenous genes was tested with Genorm software [51 (link)] and finally, ACTB and B2M genes were employed for normalization. Data for primers (designed using Primer Select software; DNASTAR, Madison, WI, USA), amplicon lengths and PCR efficiencies are indicated inTable 6 .
The qPCR assessment included genes involved in antioxidant homeostasis, vascularization and prenatal growth (SOD1, CAT, HIF1A, VEGFA, NOS2, IGF1 and UCP2) and it was performed as previously described for our group [50 (link)]. Stability of endogenous genes was tested with Genorm software [51 (link)] and finally, ACTB and B2M genes were employed for normalization. Data for primers (designed using Primer Select software; DNASTAR, Madison, WI, USA), amplicon lengths and PCR efficiencies are indicated in
4
RNA-seq Analysis of BRD8 and TIP60 Knockdown
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HCT116 and SW480 cells were transfected with control siRNA, BRD8 siRNAs (#6 and #36), or TIP60 siRNAs (#2 and #11) at a concentration of 10 nM using Lipofectamine RNAiMAX (Thermo Fisher Scientific), and maintained for additional 48 h. Total RNA was extracted from the cells using the RNeasy Plus Mini kit (Qiagen, Valencia, CA, USA), and quality of RNA was assessed using the Agilent Bioanalyzer device (Agilent Technologies, Santa Clara, CA, USA). All samples that have a RIN (RNA Integrity Number) value of more than 9.0 were subjected to library preparation. RNA-seq libraries were prepared with 100 ng of total RNA using an NEBNext Ultra II Directional RNA Library Prep Kit (New England Biolabs, Ipswich, MA, USA) according to manufacturer’s instructions. The libraries were sequenced with 60 bp single-end reads to a depth of at least more than 10 million reads per sample on the HiSeq2500 platform (Illumina, San Diego, CA, USA). Sequencing data were analyzed by a standard RNA-seq analytical pipeline. Briefly, the sequencing data were aligned to human genome (hg38) using STAR aligner (v2.7.3a).51 (link) RSEM (v1.3.3)52 (link) was used to obtain the raw gene counts from the read alignments. The DESeq2 package (v1.26.0)53 (link) was used to normalize the read count data and test for differential gene expression.
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