title: GSM9344138: Batch4_Unstimulated; Homo sapiens; RNA-Seq
library_construction_protocol: RNA-containing lysates were generated directly from permeabilized HEK293T cells using the MERCURIUS DRUG-seq (BRB-seq) kit (Alithea Genomics). Cells were incubated on ice for 15 minutes in cell lysis buffer with periodic agitation, followed by centrifugation to clear debris. Lysates containing polyadenylated RNA were used immediately for barcoded reverse transcription. BRB-seq libraries were prepared using the MERCURIUS DRUG-seq kit (Alithea Genomics) according to manufacturer's instructions. Individual wells were reverse-transcribed using barcoded oligo-dT primers, enabling assignment of each sample to a unique 14 bp cell barcode and 14 bp UMI. cDNA from all wells on each plate (up to 95 barcoded samples) was pooled and purified using the Zymo Clean & Concentration Kit (Zymo Research). Residual primers were removed by exonuclease digestion, followed by second-strand synthesis. Twenty nanograms of double-stranded cDNA per plate were tagmented, indexed using Illumina-compatible adapters, and PCR-amplified. Final libraries were sequenced on an Illumina NovaSeq platform at approximately 5 million raw reads per well.