title: GSM9444036: EAC, Fresh-frozen, Sample 6; Homo sapiens; RNA-Seq
library_construction_protocol: Clinical-grade frozen esophageal adenocarcinoma (EAC) tissue samples were collected under Oxford Radcliffe Biobank-approved study 21/A093 with informed patient consent. To enable tissue-sparing single-nucleus RNA sequencing (snRNA-seq), nuclei were isolated using an optimized salt-tris (ST)-based extraction protocol adapted for EAC specimens. Frozen tissues embedded in OCT were cryosectioned at –20°C using a Leica CM1950 cryostat, generating 40–60 scrolls of 20 μm thickness per sample. Processing steps were performed on ice using pre-chilled buffers. OCT was removed by PBS washes, followed by mechanical dissociation in ST buffer supplemented with Tween-20 and BSA. Homogenates were quenched with RNase inhibitor, sequentially filtered, and centrifuged to enrich nuclei. Final nuclear suspensions were stained with Hoechst and quantified by fluorescence microscopy prior to downstream snRNA-seq library preparation. Approximately 14,000 nuclei were loaded per sample onto a Chromium X instrument (10x Genomics), with the exception of sample #5 for which ~7,600 nuclei were used. Nuclei were suspended in ST buffer and processed using the Chromium Next GEM Single Cell 5′ Kit v2 following the manufacturer's protocol. Gel bead-in-emulsions (GEMs) underwent reverse transcription, Dynabeads-based cleanup, and cDNA amplification, with one additional PCR cycle included (14 total cycles) to account for reduced RNA content in nuclei. Gene expression (GEX) libraries were constructed using the 10x Genomics Library Construction and Dual Index kits according to the user guide. cDNA and final library fragment size distributions were assessed using an Agilent TapeStation 2200, and library concentrations were quantified using a Qubit fluorometer.  Final GEX libraries were sequenced on an Illumina NovaSeq X Plus platform using a paired-end 150 bp configuration, achieving ≥20,000 read pairs per nucleus. Sequencing was performed with a 26–10–10–90 cycle setup for read 1, i7 index, i5 index, and read 2, respectively, with a 2% PhiX spike-in included as an internal sequencing control.