|
| Status |
Public on Oct 09, 2019 |
| Title |
86_S60_smartseq2_scRNA-seq |
| Sample type |
SRA |
| |
|
| Source name |
FoxD3_Nc1_Citrine+ neural crest isolated by FACS
|
| Organism |
Gallus gallus |
| Characteristics |
development: stage 5-8ss
tissue: neural crest
isolation_method: FAC sorted for FoxD3_NC1_Citrine+ cells
amplification: SMART-seqTMv4
|
| Treatment protocol |
Dissected cranial regions from electroporated embryos were dissociated with dispase (1.5mg/ml in DMEM/10mM Hepes pH 7.5) at 37oC for 15min with intermittent pipetting to achieve a single cell suspension and 0.05% Trypsin at 37oC for a final 3min. The reaction was stopped and cells were resuspended in an excess of Hanks buffer. Cells were centrifuged at 500g for 10min and resuspended in Hanks buffer, passed through a 0.22mm filter and centrifuged at 750g for 10min, pelleted cells were resuspended in 500µl Hanks buffer. Fluorescent positive cells were sorted and collected using BD FACS-Aria Fusion. We collected ~300 and ~600 NC cells per embryo at 5-6ss and 8-10ss respectively.
|
| Growth protocol |
Fertilised wild-type chicken eggs were obtained from Henry Stewart & Co (Norfolk), staged according to Hamburger and Hamilton (1951) (24). All experiments were performed on chicken embryos younger than 12 days of development, and as such were not regulated by the Animals (Scientific Procedures) Act 1986.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Individual NC cells were collected by FACS, cDNA was generated and sequencing libraries were prepared as previously described (Picelli, Faridani et al. 2014). Libraries were sequenced using 50bp single-end reads for 96 cells on the Illumina NextSeq500 platform. A 4x107 dilution of ERCC spike in control was used.
SMARTseq2
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Description |
chicken-7-8ss-counts.txt
|
| Data processing |
Reads were mapped using RNA-STAR (2.4.2a) (Dobin, Davis et al. 2013) to the chicken genome Galgal4 assembly and the ERCC spike-in controls using the default parameters. Gene expression levels were quantified as read counts using the featureCounts function from the Subread package with default parameters. Reads that aligned to more than one locus as well as ambiguous fragments were excluded from all further analysis. To remove cells with low quality sequencing, cells with a) less than 100,000 sequenced reads or b) less than 50% uniquely mapped reads were excluded from any further analysis. Further filtering was done based on the distributions of a) the number of expressed genes per cell, b) the proportion of reads on mitochondrial genes or c) the proportion of reads on ERCC spike-ins, requiring that the cells are within 3 MADs (Median Absolute Deviation) for each distribution (McCarthy, Campbell et al. 2017). In the remaining 124 cells, 10,291 genes were detected using the following criteria: a) they had any reads in more than 5 cells and b) had an average number of counts above 1. The cell-based factors from the deconvolved pool-based size factors were used for the normalisation of the gene counts, as described in (Lun, Bach et al. 2016), using pool sizes of 10, 20, 40 and 60 cells. The clustering was done with the pagoda2 package (https://github.com/hms-dbmi/pagoda2) using the infomap and the walktrap community methods. There was a 96% concordance between the two methods and the infomap method was used to obtain the final clusters. An analysis for differentially expressed genes was performed using the SCDE package (Kharchenko, Silberstein et al. 2014) to find cluster specific gene markers. For each cluster a one-versus-one approach was applied between all pairs of clusters using the default parameters.
Genome_build: galGal4
Supplementary_files_format_and_content: chicken-7-8ss-counts.txt
Supplementary_files_format_and_content: chicken-single-cell.counts.txt
|
| |
|
| Submission date |
Apr 30, 2019 |
| Last update date |
Oct 09, 2019 |
| Contact name |
Tatjana Sauka-Spengler |
| E-mail(s) |
tatjana.sauka-spengler@imm.ox.ac.uk
|
| Organization name |
MRC Weatherall Institute of Molecular Medicine
|
| Department |
University of Oxford
|
| Lab |
Sauka-Spengler lab
|
| Street address |
Radcliffe Department of Medicine
|
| City |
Oxford |
| State/province |
Oxfordshire |
| ZIP/Postal code |
OX3 9DS |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL19787 |
| Series (2) |
| GSE121527 |
Reconstruction of the global neural crest gene regulatory network in vivo |
| GSE130500 |
Reconstruction of the global neural crest gene regulatory network in vivo [RNA-seq] |
|
| Relations |
| BioSample |
SAMN11539137 |
| SRA |
SRX5771200 |
