GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM3174432

Query DataSets for GSM3174432

Status

Public on Apr 23, 2019

Title

Time course_Sen3_N708_N503

Sample type

SRA

Source name

IMR90

Organism

Homo sapiens

Characteristics

tissue: IMR90
protocol used: Smart-seq2

Treatment protocol

(Smart-seq2 Time course experiment): ER:RasG12V -expressing cells were induced into senescence for 7 days with 4-hydroxytamoxifen (4-OHT) and were isolated as single cells using FACS. Single cells that were not induced with 4-OHT were used as control. Single cells were also collected at another two time points, day 2 and day 4, before they became fully senescent on day 7. (10x Co-culture experiment): GFP cells are co-cultured with ER:Ras expressing cells and were induced into senescence with 4-OHT. Single cell 3' gene expression was generated using 10x Genomics Chromium. (Smart-seq2 Hepatocyte experiment): Ex vivo primary hepatocytes were isolated using a modified retrograde perfusion technique as previously described[27]. Briefly, following perfusion with Perfusion Medium (Gibco) for 5 min, and Liver Digest Medium (Gibco) for 10 min, the liver was excised and the capsule disrupted to yield a cell suspension that was collected in Williams' E Medium supplemented with 2% FCS. Hepatocytes were purified by pelleting through a 40% (v:v) percoll gradient prior to FACS analysis.

Extracted molecule

total RNA

Extraction protocol

Single-cell RNA-seq was performed using Smart-seq2 or 10x chromium as indicated by each experiemnt
Smart-seq2 or 10x Genomics 3' expression protocol

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

Single cell mRNA
Not included in downstream analysis

Data processing

(Smart-seq2 time course experiment):Paired-end reads were quality trimmed using Trim galore and subsequently aligned to the human reference genome, hg19, and ERCC spike-in sequences using HISAT2. Cells that have less than 200,000 hg19 aligned reads, and a ratio of ERCC RNA spike-in control aligned reads to total aligned reads that is greater than 0.5 were filtered out. 232 IMR90 cells (100 growing cells, 41 day 2 cells, 43 day 4 cells and 48 senescent cells) passed this first filtering step. Subsequently, hg19 aligned reads were randomly downsampled to 200,000 reads. Genes were quantified using HTSeq-Count[25]. Cells that have more than 80,000 total gene counts and at least 500 genes with at least one count were used for downstream analysis. 224 IMR90 cells (100 Growing cells, 41 Day2 cells, 42 Day4 cells and 41 senescent cells) passed this second filtering step. Downstream analyses were done on the 224 cells as indicated in the sample description field
(10x Co-culture experiment): Cell Ranger 2.0.1 was used to align 10x Chromium RNA-seq reads to hg19, TurboGFP and puromycin sequence from pGIPZ and neomycin sequence from pLNCX2-ER-ras_neo, using the default parameters, and to generate gene-cell matrices. The data were subsequently processed using Seurat 2.3.0 to generate t-SNE plots and three clusters were identified using sparcl 1.0.3 (https://cran.r-project.org/web/packages/sparcl/index.html).
(Smart-seq2 hepatocyte): Paired-end reads were quality trimmed using Trim galore (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and subsequently aligned to the mouse reference genome mm10 and ERCC spike-in sequences using HISAT2. The mm10 aligned reads were randomly downsampled to 50,000 and cells that have less than 50,000 reads were removed. 75 single cells from the induced and uninduced hepatocyte passed this filter. Subsequently, cells that have less than 20,000 gene count and less than 500 genes with at least one read detected were removed. 43 single cells from the induced hepatocytes and 21 cells from the uninduced hepatocytes were retained. Lastly, cells with the log-transformed number of expressed genes and library size of 3 median absolute deviation below the median value were removed [19]. 39 single cells from the induced hepatocytes and 19 cells from the uninduced hepatocytes passed these filters.
Genome_build: hg19, mm10
Supplementary_files_format_and_content: csv file contains FPKM values (Time_course_FPKM.csv) and tab-delimited files contain counts from HTSeq after downsampling (Hepatocyte_downsampled_Htseq.txt and Time_course_downsampled_HTSeq)
Supplementary_files_format_and_content: Mdm2_deleted_hepatocyte_HTSeq.txt: counts from HTSeq for cells that passed the filter (Induced_hepatocyte)
Supplementary_files_format_and_content: timecourse_HTSeq.txt: counts from HTSeq for cells that passed the filter
Supplementary_files_format_and_content: timecourse_FPKM.csv: FPKM for cells that passed the filter
Supplementary_files_format_and_content: Growing_Sen_10x_count.txt contains UMI counts from 10x dataset and Growing_Sen_10x_metadata.txt contains the metadata for the UMI counts

Submission date

Jun 04, 2018

Last update date

Apr 23, 2019

Contact name

Nicola Neretti

Organization name

Brown University

Department

Department of Molecular Biology, Cell Biology & Biochemistry

Street address

70 Ship St