biostudies-arrayexpressLoredan Stefan NiculescuHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-17152The aim of the present study was to obtain and characterize an in vitro model for endogenous APOA1 and PON1 long-time up-regulation in hepatocytes that can be further used to decipher the mechanism of their protective action. Cultured human hepatocytes (HuH-7 cell line) were transfected with CRISPR/dCas9 activation plasmids targeting APOA1/PON1 genes. Following selection with specific antibiotics, bulk RNA sequencing was used for the transcriptomic characterization of the transfected hepatocytes. Hepatocytes from human hepatocarcinoma (Huh7 line, Cell Lines Service GmbH, Germany) were cultured in RPMI-1640 supplemented with FBS (10%, v/v), penicillin (100U/mL), and streptomycin (0.1mg/mL). Huh7 cells were seeded into 12 well plates at a density of 70.000 cells/well in complete RPMI media, without antibiotics. At 70-80% confluency, the hepatocytes were transfected using the CRISPR/(d)Cas9 activation plasmids for APOA1, PON1, or Control plasmids (CP) containing resistance genes for Blasticidin, Hygromycin B and Puromycin. 1 µg DNA plasmid/mL and 2.5µL/mL transfection reagent for each transfection condition were used according to the manufacturer’s instructions (SantaCruz Biotech., USA). After 48h, the cells’ media was changed and the cells were left to recover for another 48h. After transfection, the culture media was replaced with RPMI supplemented with 10% FBS, containing selection antibiotics, Hygromycin B (50 µg/mL), Blasticidin S HCl (2 µg/mL), and Puromycin dihydrochloride (1 µg/mL). The culture media with selection antibiotics was changed every 2 days for a period of 12 days, which was sufficient for the selection antibiotics to induce the death of the un-transfected cells. After selection, the medium was replaced with antibiotics-free RPMI and the transfected and selected Huh7 were allowed to grow until confluency. Total RNA was extracted from cells using Trizol (Invitrogen, USA) based on manufacturer’s instructions. To assess RNA purity and integrity, samples were analyzed via 1% agarose gel electrophoresis, and a NanoDrop spectrophotometer was used to confirm sample purity and quantity. Bulk long-RNA sequencing (longRNA-seq) of total RNA isolated from hepatocytes was done by Novogene, UK. The analysis included an additional RNA sample quality control, directional library preparation (rRNA removal), and long-RNA sequencing on NovaSeq X Plus Series (PE150, 12G raw data per sample).biostudies-arrayexpressSample Treatment - At 70-80% confluency, the hepatocytes were transfected using the CRISPR/(d)Cas9 activation plasmids for APOA1, PON1, or Control plasmids (CP) containing resistance genes for Blasticidin, Hygromycin B and Puromycin. 1 µg DNA plasmid/mL and 2.5µL/mL transfection reagent for each transfection condition were used according to the manufacturer’s instructions (SantaCruz Biotech., USA). After 48h, the cells’ media was changed and the cells were left to recover for another 48h. After transfection, the culture media was replaced with RPMI supplemented with 10% FBS, containing selection antibiotics, Hygromycin B (50 µg/mL), Blasticidin S HCl (2 µg/mL), and Puromycin dihydrochloride (1 µg/mL). The culture media with selection antibiotics was changed every 2 days for a period of 12 days, which was sufficient for the selection antibiotics to induce the death of the un-transfected cells. After selection, the medium was replaced with antibiotics-free RPMI and the transfected and selected Huh7 were allowed to grow until confluency.Sample Collection - After CRISPR/dCas9 treatment and antibody selection of Huh7 cells, the medium was replaced with antibiotics-free RPMI and the transfected and selected Huh7 were allowed to grow until confluency. The cells were washed with PBS, then underwent RNA isolation using Trizol.Growth Protocol - Hepatocytes from human hepatocarcinoma (Huh7 line, Cell Lines Service GmbH, Germany) were cultured in RPMI-1640 supplemented with FBS (10%, v/v), penicillin (100U/mL), and streptomycin (0.1mg/mL). Huh7 cells were seeded into 12 well plates at a density of 70.000 cells/well in complete RPMI media, without antibiotics.Library Construction - Directional library preparation (rRNA removal) Sequencing libraries were generated using RNA Library Prep Kit for Illumina following manufacturer’s recommendations and index codes were added to attribute sequences to each sample.Nucleic Acid Extraction - Total RNA was extracted from cells using Trizol (Invitrogen, USA) based on manufacturer’s instructions.Sequencing - NovaSeq X Plus Series (PE150), 12 G raw data per sampleOrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesSequence Alignment - GRCh38 (Genome Reference Consortium Human Build 38) - the primary reference assembly for the human genomeData Transformation - Mapping the clean reads to the reference genome or the transcriptome is the basis for the next following analysis. We use HISAT2 to accomplish the mapping. If only the differential expression significant genes are needed, we map reads to the transcriptome directly. If the study of alternative splicing, variation, the fusion genes are needed, the alignment are carried out at the genome level. In order to avoid losing a lot of effective junction reads, we use HISAT2 software to alignment for RNA-seq sequencing data analysis and the junction reads can be positioned precisely. Gene/transcript expression level is measured by transcript abundance. The greater the abundance, the higher is the gene/transcript expression level. In our RNA-seq analysis, the gene expression level is estimated by counting the reads that map to genes or exons. Read count is not only proportional to the actual gene expression level, but is also proportional to the gene length and the sequencing depth. In order for the gene expression levels estimated from different genes and experiments to be comparable, the FPKM is used. In RNA-seq, FPKM, short for the expected number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced, is the most common method of estimating gene expression levels, which takes into account the effects of both sequencing depth and gene length on counting of fragments.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsHISAT2, TOPHAT2Illumina NovaSeq XRNA-seq of total RNAHomo sapiensCamelia Sorina StancuLoredan Stefan NiculescuJessica Issabela Catalina HaratauLaura TomaTeodora BarbalatafalseRNA-seq analysis of human hepatocyte Huh7 cells transfected with CRISPR/(d)Cas9 plasmids for transcriptional activation of APOA1 and PON1The aim of the present study was to obtain and characterize an in vitro model for endogenous APOA1 and PON1 long-time up-regulation in hepatocytes that can be further used to decipher the mechanism of their protective action. Cultured human hepatocytes (HuH-7 cell line) were transfected with CRISPR/dCas9 activation plasmids targeting APOA1/PON1 genes. Following selection with specific antibiotics, bulk RNA sequencing was used for the transcriptomic characterization of the transfected hepatocytes. Hepatocytes from human hepatocarcinoma (Huh7 line, Cell Lines Service GmbH, Germany) were cultured in RPMI-1640 supplemented with FBS (10%, v/v), penicillin (100U/mL), and streptomycin (0.1mg/mL). Huh7 cells were seeded into 12 well plates at a density of 70.000 cells/well in complete RPMI media, without antibiotics. At 70-80% confluency, the hepatocytes were transfected using the CRISPR/(d)Cas9 activation plasmids for APOA1, PON1, or Control plasmids (CP) containing resistance genes for Blasticidin, Hygromycin B and Puromycin. 1 µg DNA plasmid/mL and 2.5µL/mL transfection reagent for each transfection condition were used according to the manufacturer’s instructions (SantaCruz Biotech., USA). After 48h, the cells’ media was changed and the cells were left to recover for another 48h. After transfection, the culture media was replaced with RPMI supplemented with 10% FBS, containing selection antibiotics, Hygromycin B (50 µg/mL), Blasticidin S HCl (2 µg/mL), and Puromycin dihydrochloride (1 µg/mL). The culture media with selection antibiotics was changed every 2 days for a period of 12 days, which was sufficient for the selection antibiotics to induce the death of the un-transfected cells. After selection, the medium was replaced with antibiotics-free RPMI and the transfected and selected Huh7 were allowed to grow until confluency. Total RNA was extracted from cells using Trizol (Invitrogen, USA) based on manufacturer’s instructions. To assess RNA purity and integrity, samples were analyzed via 1% agarose gel electrophoresis, and a NanoDrop spectrophotometer was used to confirm sample purity and quantity. Bulk long-RNA sequencing (longRNA-seq) of total RNA isolated from hepatocytes was done by Novogene, UK. The analysis included an additional RNA sample quality control, directional library preparation (rRNA removal), and long-RNA sequencing on NovaSeq X Plus Series (PE150, 12G raw data per sample).2026-07-03T00:00:00Z2026-07-03T12:33:04.967Z2026-06-12T11:21:43.272ZE-MTAB-17152ERP194978EFO_0002944EFO_0004170EFO_0009653EFO_0003789EFO_0004917EFO_0005518EFO_0003816EFO_0004184EFO_0003969