biostudies-arrayexpressOlamide AnimasahunHomo sapienshttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15689Although, there has been strong correlation between PNPLA3-I148M variants and metabolic dysfunction-associated steatotic liver disease (MASLD), the mechanism by which this variant drive the disease is not well understood. To this end, we perform this experiment to understand the transcriptomic changes that can be attributed to this variant. The total RNA was extracted from isolated primary human hepatocytes using RNeasy Plus Micro Kit (Qiagen). The sequencing library was constructed by following the Illumina Nextera XT Sample Preparation Guide. One nanogram of input cDNA was tagmented and amplified using the Illumina Nextera XT kit. Equimolar amounts of each sample were finally pooled and sequenced on an Illumina Nextseq 500 system, using a paired-end 75-bp strategybiostudies-arrayexpressLibrary Construction - The sequencing library was constructed by following the Illumina Nextera XT Sample Preparation Guide.Nucleic Acid Extraction - RNA integrity was assessed using the High Sensitivity RNA ScreenTape system on an Agilent 2200 TapeStation (Agilent). The SMART-Seq HT Kit (Takara Bio) was used to generate cDNA from 10 ng of total RNA, and the cDNA product was checked by an Agilent Fragment Analyzer system (Agilent) for quality control.Sequencing - One nanogram of input cDNA was tagmented and amplified using the Illumina Nextera XT kit. Equimolar amounts of each sample were finally pooled and sequenced on an Illumina Nextseq 500 system, using a paired-end 75-bp strategySample Collection - total RNA was extracted from isolated primary human hepatocytes using RNeasy Plus Micro Kit (Qiagen).OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - The quality of the raw fastq files were checked using FASTQC (version 0.11.9). In summary, the FastQC reports shows that the guanine-cytosine content of the samples ranges from 47% to 54%. As reference, a guanine-cytosine content between 30-40% is considered as too low as the DNA will be unstable, while a guanine-cytosine content between 70 -80% is considered to be too high because it makes PCR amplification more difficult. So, a guanine-cytosine content of about 50 to 60% is desirable10. Another important metric is the mean quality scores. All the samples have a score that falls within the acceptable threshold of 30. The QC-passed raw reads were then aligned to genome with STAR (version 2.7.5a)11. The quality of the aligned reads were assessed using QoRTs (version 1.3.6)12. The quality of the aligned reads was assessed based on the percentage of novel splice events. For all the samples, the percentage of novel splice events is about 1%. We are not expecting too many novel splicing events in this set of samples, therefore, this value is within acceptable limits. Also, the sample pass the strandedness test. For all the samples, about 97% of the reads are mapped to the first strand. This attests to the quality of the sequenced RNA fragments. Downstream analysis were performed using DESeq2 in R (CRAN 4.04), BEAVR (a Browser-based tool for the Exploration And Visualization of RNAseq data)13, GSEA14, 15 and GraphPad prism version 8. The p value was computed based on Wald test statistics. Genes with fold change greater than 1.5 and p-value less than 0.05 are considered significant.UnknownTranscriptomicsGenomicsProteomicsNextSeq 500RNA-seq of total RNAHomo sapiensPNPLA3-I148M genetic variant rewires lipid metabolism to drive programmed cell death in human hepatocytesRodrigo M Florentino, Olamide Animasahun, Nils Haep, Minal Nenwani, Kehinde Omoloja, Leyla Nurcihan Altay, Abhinav Achreja, Kazutoyo Morita, Takashi Motomura, Ricardo Diaz-Aragon, Lanuza AP Faccioli, Yiyue Sun, Zhenghao Liu, Zhiping Hu, Bo Yang, Fulei Wuchu, Ajay Shankaran, Miya Paserba, Annalisa M Baratta, Shohrat Arazov, Zehra N Kocas-Kilicarslan, Noah Meurs, Jaideep Behari, Edgar N Tafaleng, Jonathan Franks, Alina Ostrowska, Takahiro Tomiyama, Kyohei Yugawa, Akinari Morinaga, Zi Wang, Kazuki Takeishi, Dillon C. Gavlock, Mark Miedel, D Lansing Taylor, Ira J Fox, Tomoharu Yoshizumi, Deepak Nagrath , Alejandro Soto-GutierrezOlamide AnimasahunfalseRNA-Seq of isolated primary human hepatocytes carrying the PNPLA3-I148M mutation compared to the WTAlthough, there has been strong correlation between PNPLA3-I148M variants and metabolic dysfunction-associated steatotic liver disease (MASLD), the mechanism by which this variant drive the disease is not well understood. To this end, we perform this experiment to understand the transcriptomic changes that can be attributed to this variant. The total RNA was extracted from isolated primary human hepatocytes using RNeasy Plus Micro Kit (Qiagen). The sequencing library was constructed by following the Illumina Nextera XT Sample Preparation Guide. One nanogram of input cDNA was tagmented and amplified using the Illumina Nextera XT kit. Equimolar amounts of each sample were finally pooled and sequenced on an Illumina Nextseq 500 system, using a paired-end 75-bp strategy2025-10-14T00:00:00Z2025-10-14T16:33:29.66Z2025-10-09T16:45:15.657ZE-MTAB-15689ERP181291EFO_0002944EFO_0004170EFO_0009653EFO_0005518EFO_0003816EFO_0004184