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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Sep 21, 2010 |
| Title |
START (ES) L013 |
| Sample type |
SRA |
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| Source name |
ES R1
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| Organism |
Mus musculus |
| Characteristics |
cell type: embryonic stem cell-derived spheres (ES R1)
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| Growth protocol |
ES R1 cells were cultured as previously described (Moliner, A., Enfors, P., Ibanez, C.F. & Andang, M. Mouse embryonic stem cell-derived spheres with distinct neurogenic potentials. Stem Cells Dev 17, 233-243 (2008)). MEFs and Neuro-2A cells were grown in DMEM with 10% FBS, 1x penicillin/streptomycin, 1x Glutamax and 0.05 mM 2-mercaptoethanol.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were dissociated enzymatically using TrypLE Express (Invitrogen), washed and resuspended in phosphate-buffered saline (PBS). A single cell was collected into each well of a 96-well capture plate (AbGene Thermo-Fast 96 cat. No. 0600) by fluorescence-activated cell sorting (FACS), and the plate was immediately frozen on dry ice.
The cell capture plate contained a single cell per well in 5 µL of START buffer (20 mM Tris-HCl pH 8.0, 75 mM KCl, 6 mM MgCl2, 0.02% Tween-20) with 400 nM STRT-T30-BIO (5'-biotin-AAGCAGTGGTATCAACGCAGAGT30VN-3'; this and all other oligos were from Eurofins MWG Operon) and 400 nM STRT-FW-n (5'-AAGCAGTGGTATCAACGCAGAGTGGATGCTXXXXXrGrGrG-3', where “rG” denotes a riboguanine and “XXXXX” was a 5 bp barcode). Each well of the capture plate contained a different template-switching helper oligo (STRT-FW-n) with a distinct barcode.
To purify the cDNA and remove unreacted primers, 50 µL PB (Qiaquick PCR Purification Kit, Qiagen) was added to each well. All 96 reactions were pooled and purified over a single Qiaquick column. The cDNA was eluted in 30 µL EB in a 1.5 mL polyallomer tube (Beckman).
The whole 96-cell cDNA sample was amplified in a single tube in 100 µL of 200 µM dNTP, 200 µM STRT-PCR primer (5'-biotin-AAGCAGTGGTATCAACGCAGAGT-3’; Eurofins MWG Operon), 1x Advantage2 DNA Polymerase Mix (Clontech) in 1x Advantage2 PCR buffer (Clontech) with 1 min at 94°C followed by 25 cycles of 15 s at 95°C, 30 s at 65°C, 3 min at 68°C, with heated lid. An aliquot was visualized on a 1.2% agarose E-gel (Invitrogen) and the sample was amplified an additional 1-5 cycles if necessary.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Single-cell tagged reverse transcription (START) on 96 ES R1 cells
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| Data processing |
Matrix: Raw reads were sorted by barcode, trimmed to remove the barcode and any leading G or trailing A tracts, then mapped to the mouse genome using Bowtie with the default settings. Unmapped reads were discarded. Then, for each annotated feature in the NCBI 37.1 assembly, all mapping reads were counted to generate a raw count. Finally, the raw reads for each cell were normalized to transcripts per million (t.p.m.) . The output file is a tab-delimited matrix with one column per barcode (=single cell) and one row per annotated genome feature (gene, repeat, mitochondrial gene).
Wiggle: Aligned reads, regardless of barcode, were placed on the genome and the total coverage was calculated for each nucleotide position. One wiggle file was produced for each strand (_fw.wig.gz and _rev.wig.gz).
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| Submission date |
Apr 02, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Sten Linnarsson |
| E-mail(s) |
sten.linnarsson@ki.se
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| Phone |
+46852487577
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| URL |
http://ki.se
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| Organization name |
Karolinska Institutet
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| Department |
MBB
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| Lab |
Molecular Neurobiology
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| Street address |
Scheeles väg 2
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| City |
Stockholm |
| ZIP/Postal code |
17177 |
| Country |
Sweden |
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| Platform ID |
GPL9250 |
| Series (1) |
| GSE21180 |
Characterization of the single-cell transcriptional landscape by highly multiplex RNA-Seq |
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| Relations |
| SRA |
SRX056815 |
| BioSample |
SAMN00259537 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM530064_L013_fw.wig.gz |
3.1 Mb |
(ftp)(http) |
WIG |
| GSM530064_L013_matrix.tab.gz |
370.3 Kb |
(ftp)(http) |
TAB |
| GSM530064_L013_rev.wig.gz |
2.8 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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