title: GSM8980101: WT, DDC, rep2; Mus musculus; RNA-Seq
library_construction_protocol: Nuclei were prepared from fresh-frozen mouse livers using the protocol published by Humpherys and Kirita (doi.org/10.1038/nmeth.4407; dx.doi.org/10.17504/protocols.io.nahdab6) with a modification: Addition of 50 µL of 4% paraformaldehyde to the homogenate after passing through a cell strainer (dx.doi.org/10.17504/protocols.io.bhpej5je). The single-nucleus RNA sequencing (sNuc-seq) assay was performed according to the manufacturer's instructions (Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index), 10x Genomics). Briefly, nuclei were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate a gel bead-in-emulsion. The poly-A RNA from the cell lysate contained in every gel bead-in-emulsion was reverse transcribed to cDNA, adding an Illumina R1 primer sequence, unique molecular identifiers (UMIs), and the 10x Barcode. The barcoded cDNA was then cleaned up with Silane DynaBeads and amplified for 18 cycles. Full-length, barcoded cDNA was then enzymatically fragmented, sized-selected using SPRIselect reagent, adapter-ligated, and amplified for library construction. During library construction, Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences, and sample indexes were added. Libraries were pooled and sequenced on the NovaSeq 6000 using S4 flow cells with the following sequencing parameters: R1: 28 cycles, i7: 10 cycles, i5: 10 cycles, R2: 90 cycles.