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Sample GSM1599499 Query DataSets for GSM1599499
Status Public on May 20, 2015
Title mouse ES cells LIF-, 7 days (799 cells)
Sample type SRA
Source name mouse 129/Ola strain called IB10 (subcloned from E14)
Organism Mus musculus
Characteristics cell type: embryonic stem cell (ES)
days post-lif: 7
number cells in sample: 799
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.
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.
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).
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NextSeq 500
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.
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 GPL19057
Series (1)
GSE65525 Droplet barcoding for single cell transcriptomics applied to embryonic stem cells
BioSample SAMN03324852
SRA SRX863256

Supplementary file Size Download File type/resource
GSM1599499_ES_d7_LIFminus.csv.bz2 5.2 Mb (ftp)(http) CSV
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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