title: TD22_HLC_Rep3_2_Medium_8hr_H2
organism: Homo sapiens
description: Protocols: After 8 and 24 hours post-exposure, cells across all conditions were 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. hiPSC-derived HLCs were generated following a 40-day differentiation protocol. hiPSCs were seeded at 8,421 cells per well in 96-well plates (Greiner Bio-One, Cat #655161). After 24 hours, the medium was replaced with mTESR1 lacking 1x RevitaCell Supplement. Differentiation was initiated 48–72 hours later, once cells reached 70% confluency, using liver differentiation medium (LDM) supplemented with defined cytokines. From day 0 to 10, the medium was changed every other day and contained 0.6% DMSO. Starting on day 4, 5 µg/mL Doxycycline (Selleckchem, Cat#S5159) was added to induce HC3X expression. On day 12, DMSO was raised to 2%, and both non-essential and essential amino acids (Gibco, Cat#11140050 and Cat#11130051) were added to create LDM-AA. From day 14 to 50, the medium was further supplemented with 20 g/L glycine (LDM-AAGLY), and DMSO was removed. By day 50, cells were ready for exposure experiments. All growth factors were sourced from PeproTech. Prior to exposure, 500x stock solutions of Nitrofurantoin (Merck, N7878) were prepared in DMSO. On day 50, HLCs were exposed by adding 50 µL of LDM-AAGly medium containing 4.5 µg/mL doxycycline and 20 ng/mL HGF to wells already containing 50 µL of differentiation medium. All exposures were performed in triplicate, with both biological and technical replicates, using independent compound stock preparations. 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.