title: GSM8770204: WT1 scATAC-seq; Mus musculus; OTHER
library_construction_protocol: Mice were euthanized by isoflurane anesthesia followed by decapitation, with four biological replicates per group. Livers were dissected quickly after euthanasia, briefly washed in ice-cold 1X PBS, snap-frozen in liquid nitrogen, and stored at -80ºC until nuclei purification. Flash-frozen mouse liver tissue samples were processed to isolate nuclei using a combination of lysis, purification, and permeabilization steps. Nuclei were prepared by homogenizing the tissue in a lysis buffer, followed by filtration and resuspension in diluted nuclei buffer optimized for subsequent steps. This buffer formulation, containing magnesium and RNase inhibitors, ensured compatibility with downstream transposition and barcoding processes. Purified nuclei were subjected to transposition in a reaction mix containing transposase, which preferentially fragmented open-chromatin regions and appended adapter sequences to the ends of DNA fragments. Polyadenylated RNA molecules within the nuclei were reverse-transcribed into full-length cDNA. These processes occurred within nanoliter-scale Gel Beads-in-emulsion (GEMs), generated using the Chromium Next GEM platform. Each GEM contained the necessary reagents for DNA and RNA barcoding, ensuring unique molecular and nucleus-specific indexing. Following GEM incubation, barcoded DNA and cDNA were recovered, purified, and amplified via PCR to generate sufficient material for library construction. ATAC libraries underwent size selection, while cDNA for gene expression libraries was enzymatically fragmented, end-repaired, and adapter-ligated prior to further amplification. Both libraries were subjected to QC to ensure high fidelity. Sequencing of the final libraries was performed on an Illumina NovaSeq platform using paired-end 150 bp reads (PE150), targeting a depth of ~200 million reads per library.
identifiers.namespace: GEO
identifiers.id: GSM8770204_r1