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| Status |
Public on Sep 27, 2018 |
| Title |
axo2.D6 |
| Sample type |
SRA |
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| Source name |
Stage 44 forelimb limb bud
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| Organism |
Ambystoma mexicanum |
| Characteristics |
sc-rna-seq method: Fluidigm C1
development stage: stage 44 limb bud
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| Treatment protocol |
no connective tissue enrichment
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| Extracted molecule |
total RNA |
| Extraction protocol |
Single cell isolation and cDNA generation using Fluidigm C1 platform with reagents provided by Fluidigm as well as the SMARTer v2 Ultra Low RNA kit for Illumina (Clontech). External RNA spike-in transcripts (ERCC spike-in Mix, Ambion) were added to all single cell lysis reactions at a dilution of 1:40,000.
Libraries were prepared using Illumina Nextera XT kit according to illumina's 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 |
Table_S7.csv
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| Data processing |
"freeIbis" was used for C1 and SmartSeq2 sample data as an efficient basecaller with calibrated quality scores for Illumina sequencers. (Bioinformatics. 2013 May 1;29(9):1208-9. doi: 10.1093/bioinformatics/btt117. Epub 2013 Mar 6.) Cellranger, version 2.0.0, as part of the 10x Genomics analysis pipeline was used to call bases for 10x experiments.
"leeHom" was used to trim adapters and merge Illumina sequencing reads for C1 and SmartSeq2 sample data. (Nucleic Acids Res. 2014 Oct;42(18):e141. doi: 10.1093/nar/gku699. Epub 2014 Aug 6.) Cellranger, version 2.0.0, of the 10x Genomics analysis pipeline was used to trim adapters for 10x experiments.
"deML" was used for robust demultiplexing of Illumina sequences for C1 and SmartSeq2 sample data using a likelihood-based approach. (Bioinformatics. 2015 Mar 1;31(5):770-2. doi: 10.1093/bioinformatics/btu719. Epub 2014 Oct 30.) Cellranger, version 2.0.0, of the 10x Genomics analysis pipeline was used to demultiplex reads.
Reads were aligned against the axolotl transcriptome (www.axolotl-omics.org, Download: 2015-07-29) using kallisto (Nat Biotechnol. 2016 May;34(5):525-7. doi: 10.1038/nbt.3519. Epub 2016 Apr 4.) for C1 and SmartSeq 2 sample data or using STAR (Bioinformatics. 2013 Jan 1;29(1):15-21. doi: 10.1093/bioinformatics/bts635. Epub 2012 Oct 25.) implemented in 10x' cell ranger software, version 2.0.0, for 10x data.
Transcript Per Million (TPM) values were computed using kallisto for C1 and SmartSeq2 samples. (Nat Biotechnol. 2016 May;34(5):525-7. doi: 10.1038/nbt.3519. Epub 2016 Apr 4.) Cellranger, version 2.0.0, implemented in the 10x Genomics analysis pipeline was used to obtain bam alignment files and gene count (UMI) matrices.
Gene expression values of different isoforms of the same gene were summed using custom Perl scripts.
Genome_build: Axolotl transcriptome (Am_2.2)
Supplementary_files_format_and_content: Master data frame including cell ID, cell type, timepoint, gene ID, and TPM values for each single cell.
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| Submission date |
Jun 19, 2018 |
| Last update date |
Sep 27, 2018 |
| Contact name |
Tobias Gerber |
| E-mail(s) |
tobias.gerber@embl.de
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| Organization name |
EMBL Heidelberg
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| Department |
Developmental Biology
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| Lab |
Detlev Arendt
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| Street address |
Meyerhofstr. 1
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| City |
Heidelberg |
| ZIP/Postal code |
69117 |
| Country |
Germany |
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| Platform ID |
GPL22800 |
| Series (1) |
| GSE106269 |
Single-cell analysis uncovers convergence of cell identities during axolotl limb regeneration |
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| Relations |
| BioSample |
SAMN09458405 |
| SRA |
SRX4241066 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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