Illumina NovaSeq 6000 paired end sequencing; Investigating NAFLD from RNA-seq data (human cell line Huh7) for samples under three treatments: low fat low sugar, low fat high sugar and high fat high sugar. Wild type samples are compared against DGAT2 knocked out.
Investigating NAFLD from RNA-seq data (human cell line Huh7) for samples under three treatments: low fat low sugar, low fat high sugar and high fat high sugar. Wild type samples are compared against DGAT2 knocked out.
For this experiment, Human hepatocellular carcinoma cells (Huh7) were used. After passaging, cells were seeded and grown to confluence in maintenance media, before being cultured in human serum (HS)-containing media for seven days. HS media consisted of phenol red-free DMEM, 11 mmol/L glucose, 1% NEAAs, 10,000 U/mL penicillin-streptomycin, 1% sodium pyruvate and 1% GlutamaxTM, with 2% HS. Cells were treated with one of three experimental treatments. All treatments consisted of HS media described in the growth protocol with a combination of supplementary fatty acids (FAs) and fructose. Low fat low sugar (LFLS) media had an additional 200 μM fatty acids (FAs), low fat high sugar (LFHS) media had 200 μM FAs and 5.5 mM fructose added and the high fat high sugar (HFHS) media had 800 μM FAs and 5.5 mM fructose added. FAs consisted of a physiological mix of oleic acid, palmitic acid, linolenic acid and α-linoleic acid at a ratio of 45:30:24:1. FAs were first dissolved in ethanol before being conjugated to BSA at a stock concentration of 10 mM FAs to 10% (w/v) BSA and diluted to 800 μM or 200 μM before treatment. For assessment of de novo lipogenesis (DNL), 10% heavy water (2H2O) was added to media and for all experiments, media was changed every 48–72 h. RNA was extracted using the RNEasy® Plus mini kit according to manufacturer's instructions. The resulting RNA was treated with DNase (QIAGEN) and quality checked with an Agilent Bioanalyzer. Samples with RIN values higher than 7 (24 out of an initial number of 28 samples) were used to construct Illumina libraries. The NEB Next® Ultra RNA Library Prep Kit is used for the library preparation. After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads. The mRNA is fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) is added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick translation. The final cDNA library is ready after a round of purification, terminal repair, A-tailing, ligation of sequencing adapters, size selection and PCR enrichment. Quality control test for the library construction follows three steps:(1) Qubit 2.0: tests the library concentration preliminarily; (2) Agilent 2100: tests the insert size; (3) Q-PCR: quantifies the library effective concentration precisely.