title: GSM6112228: P75-NASH with cirrhosis-D-SITTE5; Homo sapiens; RNA-Seq
library_construction_protocol: Tissue lysis by dounce homogenisation and nuclei isolation by flow cytometry using scatter properties and DAPI staining to label nuclei. Single-nuclei RNA-seq libraries were prepared using the following: Chromium Single Cell 3′ Library & Gel Bead Kit v3.1, Chromium Chip G Kit and Chromium Single Cell 3' Reagent Kits v3.1 User Guide (Manual Part CG000316 Rev A; 10X Genomics). For each sample 16k nuclei were loaded on the Chromium instrument with the expectation of collecting gel-beads emulsions containing nuclei cells. RNA from the barcoded nuclei for each sample was subsequently reverse-transcribed in a C1000 Touch Thermal cycler (Bio-Rad) and all subsequent steps to generate single-nuclei libraries were performed according to the manufacturer's protocol with 19 PCR cycles in cDNA amplification step. cDNA quality and quantity was measured with Agilent TapeStation 4200 (High Sensitivity 5000 ScreenTape) after which 25% of material was used for gene expression library preparation. Library quality was confirmed with Agilent TapeStation 4200 (High Sensitivity D1000 ScreenTape to evaluate library sizes) and Qubit 4.0 Flourometer  (ThermoFisher Qubit™ dsDNA HS Assay Kit to evaluate dsDNA quantity). Each sample was normalized and pooled in equal molar concentration. To confirm concentration pool was qPCRed using KAPA Library Quantification Kit on QuantStudio 6 Flex before sequencing. Pool was sequenced on Illumina NovaSeq6000 sequencer with following parameters: 28 bp, read 1; 10 bp, i5 index; 10bp, i7 index; 90 bp, read 2