|
| Status |
Public on Apr 04, 2024 |
| Title |
P75-NASH with cirrhosis-D-SITTE5 |
| Sample type |
SRA |
| |
|
| Source name |
liver biopsy
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: liver
disease status: NASH with cirrhosis
patient id: P75
liver lobe: Right
gender: M
saf score: S1A3F4
age: 60
|
| Treatment protocol |
Biopsies were snap frozen in liquid nitrogen
|
| Extracted molecule |
total RNA |
| Extraction 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
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
Quality control was peformed using fastQC ( v0.11.8) and multiQC (v 1.9)
Alignment, filtering and gene quantification was performed using 10x cellranger v5.0.0; the alignment was perfomed against the H Sapiens reference genome (GRCh38.p13)
Normalization of expression was levels was performed with SCTransform (Hafemeister and Satija, 2019)
Clustering was performed using SLM algorithm from the FindClusters function from Seurat (Satija, 2021)
Differential Expression was perfored using the Wilcoxon Rank Sum test from FindMarkers function from Seurat (Satija, 2021)
Assembly: GRCh38.p13
Supplementary files format and content: Raw and norrmalised count matrices, in csv format.
Supplementary files format and content: Seurat object, rds format.
|
| |
|
| Submission date |
May 06, 2022 |
| Last update date |
Jul 05, 2024 |
| Contact name |
Irina Mohorianu |
| E-mail(s) |
data-submissions@stemcells.cam.ac.uk
|
| Organization name |
University of Cambridge
|
| Department |
Wellcome-MRC Cambridge Stem Cell Institute
|
| Street address |
Puddicombe Way
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 0AW |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE202379 |
Single Nuclei RNA-Seq to map progression of non-alcoholic fatty liver disease (NAFLD) in patients II |
|
| Relations |
| BioSample |
SAMN28116276 |
| SRA |
SRX15196917 |
