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| Status |
Public on Dec 19, 2017 |
| Title |
HH15_Leader_4 |
| Sample type |
SRA |
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| Source name |
Cranial Neural Crest Cells
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| Organism |
Gallus gallus |
| Characteristics |
location: Leader
development stage: Hamburger & Hamilton (HH) Stage 15
type: In Vivo
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| Treatment protocol |
For NC cell isolations, pre-migratory NC were labeled by electroporation of a Gap43-YFP plasmid DNA into the dorsal neural tube of HHSt9 embryos as previously described (McLennan and Kulesa, 2007) and re-incubated (8, 16 and 24 hr for HHSt11, 13 and 15, respectively). After re-incubation, embryos were harvested into chilled 0.1% DEPC PBS and screened for health and transfection efficiency before tissue isolations were performed.
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| Growth protocol |
Fertilized, white leghorn chicken eggs were incubated in a humidified chamber at 38C to the developmental desired stage. For additional information, please see methods in PMID: 29199959 DOI: 10.7554/eLife.28415
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
For scRNA-seq, the NC r4 stream was isolated by manual dissection at HHSt11, 13 and 15. HHSt11 r4 streams containing few migrating NC were pooled. For HHSt13 and 15 embryos, the r4 NC stream was further subdivided by manual dissection into three spatially distinct, non-overlapping portions: the most invasive front (5% of the stream, termed ‘Front’), the leading portion of the stream (25% of stream proximal of the most invasive front, termed ‘Lead’) and the proximal-most trailing portion of the stream (70% of the stream, termed ‘Trail’). Developmental stage- and stream position-matched tissues were pooled and dissociated before cytometric isolation. Cells were isolated by FACS, which included forward scatter, side scatter, pulse width, live/dead stain and YFP gates. Cells were sorted directly into 6 ul of Clontech lysis solution containing 0.05% RNAse inhibitor. Following lysis for 5 min at room temperature, lysates were immediately frozen on dry ice and stored at −80C.
Single-cell lysates were thawed on ice. cDNA synthesis and library preparation were performed with SMART-seq v4 Ultra Low Input RNA-seq (634892, Takara, Kusatsu, Shiga, Japan) and Nextera XT DNA dual indexing library preparation kits as recommended by the manufacturer (FC-131–1096, Illumina). Despite more cost-effective scRNAseq strategies, SMARTseq v4 was employed in order to detect the greatest number of genes per single cell, which is critical for distinguishing highly similar cell subpopulations (Ziegenhain et al., 2017; Dueck et al., 2016).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
single cells isolated by FACS
rpkms_sc.txt
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| Data processing |
Fastq files were generated with bcl2fastq2 (2.18.0.12).
Reads were aligned to the chicken genome galGal4 from UCSC using Tophat 2.0.13 with -x 1 -g 1. Annotations were from Ensembl 80. Counts of reads on genes were calculated using bedtools intersect on unioned exons.
Data was read into R and differentially expressed genes were found using edgeR with TMM normalization.
Genome_build: galGal4
Supplementary_files_format_and_content: tab delimited text of TMM normalized, edgeR generated rpkms.
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| Submission date |
Dec 18, 2017 |
| Last update date |
Dec 19, 2017 |
| Contact name |
Madelaine Gogol |
| Organization name |
Stowers Institute
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| Department |
Computational Biology Core
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| Street address |
1000 E. 50th Street
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| City |
Kansas City |
| State/province |
MO |
| ZIP/Postal code |
64110 |
| Country |
USA |
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| Platform ID |
GPL19005 |
| Series (2) |
| GSE108221 |
Single-cell transcriptome analysis of avian neural crest migration reveals signatures of invasion and molecular transitions [scRNA-seq] |
| GSE108230 |
Single-cell transcriptome analysis of avian neural crest migration reveals signatures of invasion and molecular transitions |
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| Relations |
| BioSample |
SAMN08199802 |
| SRA |
SRX3480483 |
| 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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