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| Status |
Public on Apr 28, 2020 |
| Title |
R1_4Dprimed [II] |
| Sample type |
SRA |
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| Source name |
R1 embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: R1
treatment: LIF+FGF2+Activin A+IWP2 for 4 days
passage number: p24
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| Treatment protocol |
Cells were cultured in the presence of LIF and the indicated supplements for the indicated duration. 2i is CHIR99021+PD325901. Concentrations: CHIR99021 3 uM; PD325901 0.1 uM; IWP2 2 uM; FGF2 12 ng/ml; human Activin A 20 ng/ml.
After culture, the cells were trypsinized and FACS sorted into 384-wells plates containing 384 primers, dNTPs and Mineral oil (Sigma). For each sample, 192 wells were used. Wells 167, 168, 191 and 192 for each sample were left empty. After sorting, plates were snap-frozen on dry ice and stored at -80° C.
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| Growth protocol |
Cells were grown in serum-free N2B27 medium on tissue culture plastic plates coated with 0.1% gelatin and fetal calf serum.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were heat-lysed at 65° C for 5 min.
Following lysis, cDNA was synthesized using the CEL-Seq2 protocol (Hashimshony, T. et al. CEL-Seq2: sensitive highly-multiplexed single-cell RNA-Seq. Genome biology 17, 77 (2016)) and robotic liquid handling platforms. After second strand cDNA synthesis, the barcoded material was pooled into libraries of 384 cells and amplified using IVT. Following amplification, the rest of the CEL-seq2 protocol was followed for preparation of the amplified cDNA library, using TruSeq small RNA primers (Illumina).
The DNA library was paired-end sequenced on an Illumina Nextseq™ 500, high output, with a 1x75 bp Illumina kit (R1: 26 cycles, index read: 6 cycles, R2: 60 cycles).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
R1 ES cells differentiated to primed pluripotency.
processed data file: normcounts_R1_IB10_L2I_LIM_Prim.txt
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| Data processing |
During sequencing, Read 1 was assigned 26 base pairs and was used for identification of the Illumina library barcode, cel barcode and UMI. R2 was assigned 60 base pairs and used to map to the reference transcriptome of mm10 with BWA. Data demultiplexing and statistical UMI poisson counting correction was done as described in Grun, D., Kester, L. & van Oudenaarden, A. Validation of noise models for single-cell transcriptomics. Nat Methods 11, 637-640 (2014).
Mapping and generation of count tables were done using the MapAndGo script https://github.com/anna-alemany/transcriptomics/tree/master/mapandgo . The empty wells (167, 168, 191 and 192) were removed from the normalized count matrix.
Single-cell transcriptomics analysis was done using the RaceID3 algorithm, following an adapted version of the RaceID manual (https://cran.r-project.org/web/packages/RaceID/vignettes/RaceID.html). In total, 1152 cells were sequenced.
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: normcounts_R1_IB10_L2I_LIM_Prim.txt: Tab-delimited text file containing a matrix of normalized read counts.
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| Submission date |
Feb 21, 2020 |
| Last update date |
Apr 28, 2020 |
| Contact name |
Derk ten Berge |
| E-mail(s) |
d.tenberge@erasmusmc.nl
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| Phone |
+31107043452
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| Organization name |
ErasmusMC
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| Department |
Cell Biology
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| Street address |
Wytemaweg 80
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| City |
Rotterdam |
| ZIP/Postal code |
3015CN |
| Country |
Netherlands |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE145726 |
In vitro capture and characterization of embryonic rosette-stage pluripotency between naive and primed states (II, single-cell RNA-Seq data) |
| GSE145727 |
In vitro capture and characterization of embryonic rosette-stage pluripotency between naive and primed states |
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| Relations |
| BioSample |
SAMN14162157 |
| SRA |
SRX7778840 |
| 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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