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Status |
Public on Apr 23, 2019 |
Title |
Uninduced_hepatocyte_6_2c71 |
Sample type |
SRA |
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Source name |
colony N4 C57/Bl6J background
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Organism |
Mus musculus |
Characteristics |
background strain: C57/Bl6J tissue: Hepatocyte protocol used: Smart-seq2
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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.
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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
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina HiSeq 2500 |
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Description |
Single cell mRNA Not included in downstream analysis
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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
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Submission date |
Jun 04, 2018 |
Last update date |
Apr 23, 2019 |
Contact name |
Nicola Neretti |
Organization name |
Brown University
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Department |
Department of Molecular Biology, Cell Biology & Biochemistry
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Street address |
70 Ship St
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City |
Providence |
State/province |
RI |
ZIP/Postal code |
02903 |
Country |
USA |
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Platform ID |
GPL17021 |
Series (1) |
GSE115301 |
Notch signalling mediates secondary senescence |
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Relations |
BioSample |
SAMN09336541 |
SRA |
SRX4160148 |
Supplementary data files not provided |
SRA Run Selector |
Raw data are available in SRA |
Processed data are available on Series record |
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