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| Status |
Public on Apr 27, 2023 |
| Title |
Day4 (HH24) Chick Trunk_puck25_Slide-seq |
| Sample type |
SRA |
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| Source name |
embryonic trunk
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| Organism |
Gallus gallus |
| Characteristics |
tissue: embryonic trunk
cell type: tissue
breed: white leghorn
genotype: wild-type
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| Treatment protocol |
none; unlabeled, wild-type tissue
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| Growth protocol |
See PMID: 36840366 (DOI: 10.1002/dvdy.577) methods
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
To determine the spatial gene expression within chick trunk tissue at the developmental stage prior to the dorsal migration of the primary SG and axon extensions from the spinal-cord based PGNs, we harvested day 4 (HH24) unlabeled chick embryos. Fresh frozen HH24 trunk tissues were transversely cryo-sectioned to approximate single cell thickness (10um) and carefully placed on a single sequenced Slide-seq puck containing ~74,000 uniquely barcoded 10um beads. Tissue was then lysed, and the poly-A mRNA captured by poly(dT).
Tissue was then lysed, and the poly-A mRNA captured by poly(dT) for downstream cDNA synthesis, NGS library construction, and Illumina sequencing on a NextSeq 500 machine using the following paired end length: 44 bp Read1, 8 bp Index and 50 bp Read2.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
NextSeq 500 Mid Output 44 * 8 * 50
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| Data processing |
Data processing was performed using the slide-seq tools pipeline from Broad Institute (https://github.com/MacoskoLab/slideseqtools). Reads were aligned to the galGal6 (Gallus gallus) genome with annotations from from Ensembl 98, and 91% of reads aligned. Downstream analysis was performed with R 3.6.1 and the Seurat package version 3.1.4.9903 (Stuart et al., 2019; Seurat v4) for unbiased spatial analysis. In total, we detected a total of 20860 beads with around 8 million UMI counts, averaging about 381.5 umi counts per gene, and 276 mean genes per bead. The non-tissue covering area was then filtered out based on original imagings. Slide-seq data was annotated by integrating with 10x single-cell data using the label transfer function in Seurat software. Prediction scores were generated for each bead based on its original single-cell cluster annotations. Data was further analyzed and visualized using in-house generated programs using freeware through Volta and Scanpy single-cell analysis in Python.
Raw data in the form of bcl files was converted to fastq using bcl2fastq v2.20.0.422.
Fastq files were aligned to the chicken genome galGal6 from UCSC with gene annotations from Ensembl 98 using STAR (2.7.3a) as part of a version of the slide-seq tools pipeline (https://github.com/MacoskoLab/slideseq-tools) modified to run on an interactive server.
Steps of the slide-seq tools pipeline include tagging read sequences with 14 base bead barcode and 8 base UMI sequences, filtering bam based on quality score, trimming start sequence and PolyA tails, alignment using STAR, annotate with GTF, run Cmatcher to get bead barcodes with mismatches <= 1, and generate barcode-gene matrix using filtered barcodes with >10 UMIs.
After slide-seq tools, data was analyzed in R 3.6.1 using Seurat 3.2.3. Area under tissue was manually selected using gatepoints package (0.1.3). After examining the elbow plot, 40 principal components were used for downstream analysis, and a resolution of .2 was used in FindClusters to identify 8 clusters.
Assembly: galGal6
Supplementary files format and content: beadbarcodes.txt.gz - File listing the original barcodes sequenced by Broad institute determined to be on the puck with their locations
Supplementary files format and content: L42443.digital_expression.txt.gz - gzipped tab-delimited text file containing a gene expression matrix with a row for each gene and a column for each slide-seq bead or barcode. Generated by slide-seq tools pipeline.
Supplementary files format and content: ss.rds - serialized R data file containing a Seurat object with low quality or non-tissue beads removed and filtered. Used for downstream analysis.
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| Submission date |
Apr 27, 2023 |
| Last update date |
Apr 27, 2023 |
| 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 |
GPL19787 |
| Series (2) |
| GSE230776 |
Cell-Type Profiling of the Sympathetic Nervous System Using Spatial Transcriptomics and Spatial Mapping of mRNA [Slide-seq] |
| GSE230780 |
Cell-Type Profiling of the Sympathetic Nervous System Using Spatial Transcriptomics and Spatial Mapping of mRNA |
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| Relations |
| BioSample |
SAMN34406820 |
| SRA |
SRX20119532 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7234197_L42443.digital_expression.txt.gz |
8.6 Mb |
(ftp)(http) |
TXT |
| GSM7234197_beadbarcodes.txt.gz |
767.9 Kb |
(ftp)(http) |
TXT |
| GSM7234197_ss.rds.gz |
189.0 Mb |
(ftp)(http) |
RDS |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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