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Sample GSM4293553

Query DataSets for GSM4293553

Status

Public on Jul 31, 2020

Title

WT9M

Sample type

SRA

Source name

Pancreas cells

Organism

Mus musculus

Characteristics

genotype: Ptf1a-CreER, LSL-Kras-G12D, LSL-tdTomato
pti: 9 months
cell type: Pancreas cells

Extracted molecule

total RNA

Extraction protocol

The pancreas was removed and washed and the acinar cells were isolated using 10mg collagenase P and 0.2mg trypsin inhibitor live cells were isolated by using MACS dead cells removal kit (Miltenyi Biotech #130-090-101) with MACS MS columns. The stomach was removed and the cells isolated first with dissociation buffer containing 2mg collagenase P and then with dissociation buffer containing 5 mg Dispase II (Roche #D4693) at 37C.
RNA libraries were prepared for sequencing using standard Illumina protocols

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Data processing

The Illumina bcl2fastq (v1.8.3) was used to convert Illumina base call files (bcl) to FASTQ files. Alignment of FASTQs to the mm10/hg19 reference was done by Cell Ranger (v3.0, 10X Genomics). mm10/hg19 reference downloaded from 10x (v1.2.0, 10X Genomics) and a tdTomato sequence gene was added to mm10. Library constriction was performed according to standard 3’ 10x genomics kit and sequence using illumine Hi-Seq and Next-seq machine.
First analysis of the data was done using R package Seurat(v2.3.4). Raw transcript counts gene-cell matrices were filtered to remove cells with: (i) fewer than 200 transcripts, (ii) total UMI counts lower then 1000,(iii) cells with more than 5% or 10% mitochondrial genes for 10x chemistry v2 or v3, respectively. In addition, genes that expressed in less than 3 cells were removed. The gene-cell matrices were then normalized such that the number of UMIs in each cell was divided by the total UMI counts, multiplied by 10,000 and log transformed. Data was scaled using ScaleData Function from Seurat to remove unwanted sources of variation, so that cell-cycle heterogeneity or UMI counts does not contribute to PCA or downstream analysis.
Genome_build: mm10 and added tdTomato gene
Genome_build: hg19
Supplementary_files_format_and_content: All processed data merged are tab-delimited csv file from raw data of the Seurat [email protected] and can be distinguish by barcode name.
Supplementary_files_format_and_content: rows are genes, columns are cell barcodes and the values are normalized and log transformed

Submission date

Feb 03, 2020

Last update date

Jul 31, 2020

Contact name

Oren Parnas

E-mail(s)

[email protected]

Organization name

Hebrew University

Department

Immunology