title: Targeted RNA-seq (TempO-Seq) of temporal concentration responses to drug exposures in PHH and HepaRG cells
design: Targeted RNA-seq (TempO-Seq) of temporal concentration responses to drug exposures in PHH and HepaRG cells
library_name: S_72h_APAP3_Rep3_s
library_construction_protocol: Following exposure, cell cultures were washed once with DPBS without calcium and magnesium chloride (Sigma-Aldrich, #D8537). Thereafter, cells were lysed with 50 µL lysis buffer (1:1 DPBS and 2x TempO‐Seq lysis buffer, BioSpyder, mixed ex tempore) for 15 minutes at room temperature and stored at ‐80°C, sealed with an aluminium silver seal (Greiner Bio-One, #676090).  PHH (10-donor, LIVERPOOL Cryoplateable Hepatocytes; BioIVT, #X008001-P) were thawed in 25 mL Sekisui XenoTech OptiThaw Hepatocyte Kit (Tebu-bio, #K8000) and centrifuged at 100x g for 10 minutes. Pellet was resuspended in INVITROGRO CP medium (BioIVT, #Z99029) supplemented with TORPEDO antibiotics mix (BioIVT, #Z990007) and seeded at a density of 70,000 cells per well in BioCoat Collagen-I-coated 96-wells plates (Corning, #354407). After 6 hours, PHH were washed with Dulbecco's phosphate-buffered saline (DPBS) to remove dead cells. PHH were overlaid with 100 µl 0.25 mg/mL cooled Matrigel™ (BD Biosciences, #354230) in INVITROGRO HI medium (BioIVT, #Z99009) supplemented with TORPEDO antibiotics mix (BioIVT, #Z990007) to create a collagen-Matrigel™ sandwich.  Cryopreserved differentiated HepaRG® cells (Biopredic International, #HPR116-TA08) were thawed and seeded (day one) according to the manufacturer's protocol. In short, 72,000 viable cells per well were seeded in collagen-coated (Corning #354236, 0.1 mg/ml in 0.02N acetic acid) 96-well plates (Falcon® #353072) in 100 µL basal hepatic cell medium (Biopredic International, #32551) supplemented with HepaRG® Thawing/Plating/General Purpose Medium (Biopredic International, #ADD670). On the second day of culture, the medium was changed to 100 µL basal hepatic cell medium with HepaRG® Maintenance/Metabolism Medium (Biopredic International, #ADD620) and subsequently renewed on days four and seven. PHH were exposed to the compounds in 100 µL INVITROGRO HI medium (BioIVT, #Z99009) supplemented with TORPEDO antibiotics mix (BioIVT, #Z990007) 24 hours after plating. On day eight in culture, HepaRG® cells were exposed to the compounds in 100 µL basal hepatic cell medium with serum-free HepaRG® Induction Medium (Biopredic International, #ADD650). Cell lysates were analysed at BioClavis (Glasgow, UK) using TempO-Seq targeted RNA sequencing technology with the human whole-transcriptome probeset version 2.0 according to their standard protocol. In TempO-Seq, each Detector Oligo consists of a sequence complementary to an mRNA target plus a universal (i.e. same for every targeted gene) primer binding site. They anneal in immediate juxtaposition to each other on the targeted RNA template such that they can be ligated together. Ligated detector oligos are PCR-amplified using a primer set (single-plex PCR reaction, with a single primer pair for each sample) that introduces both the adaptors required for sequencing and a sample-specific barcode. The barcode sequences flank the target sequence and are inserted appropriately into the standard Illumina adaptors to permit standard dual-index sequencing of the barcodes and deconvolution of sample-specific reads from the sequencing data using the standard Illumina software. All the PCR-amplified and barcoded samples are pooled into a single library for sequencing. Sequencing reads are demultiplexed using the standard sequencing instrument software for each sample using the barcodes to give a FASTQ file for each. Cell lysates were analysed at BioClavis (Glasgow, UK) using TempO-Seq targeted RNA sequencing technology with the human whole-transcriptome probeset version 2.0 according to their standard protocol. In TempO-Seq, each Detector Oligo consists of a sequence complementary to an mRNA target plus a universal (i.e. same for every targeted gene) primer binding site. They anneal in immediate juxtaposition to each other on the targeted RNA template such that they can be ligated together. Ligated detector oligos are PCR-amplified using a primer set (single-plex PCR reaction, with a single primer pair for each sample) that introduces both the adaptors required for sequencing and a sample-specific barcode. The barcode sequences flank the target sequence and are inserted appropriately into the standard Illumina adaptors to permit standard dual-index sequencing of the barcodes and deconvolution of sample-specific reads from the sequencing data using the standard Illumina software. All the PCR-amplified and barcoded samples are pooled into a single library for sequencing. Sequencing reads are demultiplexed using the standard sequencing instrument software for each sample using the barcodes to give a FASTQ file for each.