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Status |
Public on May 20, 2015 |
Title |
mouse ES cells LIF-,4 days (682 cells) |
Sample type |
SRA |
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Source name |
mouse 129/Ola strain called IB10 (subcloned from E14)
|
Organism |
Mus musculus |
Characteristics |
cell type: embryonic stem cell (ES) days post-lif: 4 number cells in sample: 682
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Treatment protocol |
In the undifferentiated state the ESC base media was supplemented with Leukemia Inhibitory Factor (LIF) at final concentration 1000 U/mL and for unguided mES differentiation the media was without LIF. Within 2 days of LIF withdrawal the culture experienced significant morphological changes indicating the differentiation of mES cells.
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Growth protocol |
The ES cells were maintained in ESC base media inside culture flasks pre-coated with gelatin at 37°C in 5% CO2 and 60-80% humidity at density ~3 × 10^5 cells ml–1. The ESC media contained phenol red free DMEM (Gibco), supplemented with 15% (v/v) fetal bovine serum (Gibco), 2 mM L-glutamine, 1x MEM non-essential amino acids (Gibco), 1% (v/v) penicillin-streptomycin antibiotics, 110 µM b-mercaptoethanol, 100 µM sodium pyruvate.
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Extracted molecule |
polyA RNA |
Extraction protocol |
Cells were encapsulated into droplets on ice and lysed in the 4nL microfluidic droplets using a final concentration of 0.4% NP-40. Single cell lysates were subject to reverse transcription at 50°C without purification of RNA. Cells were barcoded using the db-Seq platform, which makes use of the CEL-Seq protocol for library construction (Hashimshony et al., Cell Reports 2011).
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina MiSeq |
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Data processing |
Reads were first filtered based on presence in read 1 of two sample barcode components separated by the W1 adaptor sequence (GAGTGATTGCTTGTGACGCCTT) Read 2 was then trimmed using Trimomatic (5) (version 0.30; parameters: LEADING:28 SLIDINGWINDOW:4:20 MINLEN:19). Barcodes for each read were matched against a list of the 3842 pre-determined barcodes, and errors of up to two nucleotides mismatch were corrected. Reads with a barcode separated by more than two nucleotides from the reference list were discarded. The reads were then split into barcode-specific files for mapping and UMI filtering. The trimmed reads were aligned using Bowtie (version 0.12.0, parameters: -n 1 -l 15 -e 300 -m 200 -best -strata -a) to the mouse transcriptome. The reference transcriptome was built using all annotated transcripts (extended with a 125bp poly-A tail) from the UCSC mm10 genome assembly. We used a custom Python and PySAM script to process mapped reads into counts of UMI-filtered transcripts per gene. Genome_build: mm10 for ES cells; hg19 for K562 cells. Supplementary_files_format_and_content: Columns = cells; rows = genes. Column 1 contains gene symbols. Values show unique molecular identifier (UMI)-filtered counts per cell detected in the raw data. No normalization is performed.
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Submission date |
Feb 02, 2015 |
Last update date |
May 15, 2019 |
Contact name |
Allon M Klein |
Organization name |
Harvard Medical School
|
Department |
Systems Biology
|
Street address |
200 Longwood Avenue
|
City |
Boston |
State/province |
MA |
ZIP/Postal code |
02115 |
Country |
USA |
|
|
Platform ID |
GPL16417 |
Series (1) |
GSE65525 |
Droplet barcoding for single cell transcriptomics applied to embryonic stem cells |
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Relations |
BioSample |
SAMN03324857 |
SRA |
SRX863255 |