title: PHHs_Rep9_0hrs_DMSO_0
organism: Homo sapiens
description: Protocols: 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.