Targeted RNA-seq (TempO-Seq) of human hepatocellular carcinoma cell line HepG2, primary human hepatocytes (PHHs) and human induced pluripotent stem cell derived hepatocyte like cells (HLCs) in untreated condition
All cell cultures were done in 96 well plates and washed once with DPBS without calcium and magnesium chloride (Sigma-Aldrich, #D8537). Next, 50 µL lysis buffer (1:1 DPBS and 2x TempO‐Seq lysis buffer, BioSpyder) was added to the cells for 10 minutes at 37°C. Plates were immediately sealed with an aluminum silver seal (Greiner Bio-One, Cat #676090) and stored at -80°C until shipped to BioClavis, UK. Cell lysates were shipped and processed at BioClavis (Glasgow, UK) using TempO-Seq targeted RNA sequencing technology, using the human whole-transcriptome probeset version 2.0 according to the manufacturer's standard procedure. Each Detector Oligo (DO) contains a region complementary to its specific mRNA target and a universal primer binding site shared across all targets. DOs hybridize adjacently on the RNA template, enabling their ligation into a single molecule. These ligated oligos are then PCR-amplified in a single-plex reaction using a universal primer pair, which includes both the sequencing adaptors and a unique sample barcode. The barcode sequences are positioned to flank the target region and are integrated into the standard Illumina adaptors. This facilitates dual-index sequencing for accurate sample identification and de-multiplexing. All barcoded PCR products are pooled into a single sequencing library. Sequencing reads are demultiplexed by the standard Illumina software using the sample-specific barcodes, resulting in individual FASTQ files for each sample. Cell lysates were shipped and processed at BioClavis (Glasgow, UK) using TempO-Seq targeted RNA sequencing technology, using the human whole-transcriptome probeset version 2.0 according to the manufacturer's standard procedure. Each Detector Oligo (DO) contains a region complementary to its specific mRNA target and a universal primer binding site shared across all targets. DOs hybridize adjacently on the RNA template, enabling their ligation into a single molecule. These ligated oligos are then PCR-amplified in a single-plex reaction using a universal primer pair, which includes both the sequencing adaptors and a unique sample barcode. The barcode sequences are positioned to flank the target region and are integrated into the standard Illumina adaptors. This facilitates dual-index sequencing for accurate sample identification and de-multiplexing. All barcoded PCR products are pooled into a single sequencing library. Sequencing reads are demultiplexed by the standard Illumina software using the sample-specific barcodes, resulting in individual FASTQ files for each sample.