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| Status |
Public on Jun 20, 2023 |
| Title |
tail, 21 DPA, original tail portion |
| Sample type |
SRA |
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| Source name |
tail original tail portion
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| Organism |
Anolis carolinensis |
| Characteristics |
tissue: tail (original tail portion)
age: 9-12 months old
time: 21 days post amputation
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| Extracted molecule |
total RNA |
| Extraction protocol |
Lizard tails were anesthetized by exposing tails to a spray of ethyl chloride for 10 seconds and amputated with a sterile scalpel blade to begin regeneration. Tail samples were collected in Hank’s balanced salt solution (HBSS) supplemented with 100 units/mL penicillin and 100 µg/mL streptomycin (HBSS with P/S). Samples were collected at 0, 1, 3, 7, 14, 21, and 28 days after initial amputation or days post-amputation (DPA) for cell dissociation. Longer tail samples (14, 21 and 28 DPA) were further split into two portions at the original amputation plane using a sterile scalpel. Three tail samples per time point were cut to 3-5 mm pieces in length and each piece was cut into 1/8ths. Tail pieces were added to gentleMACS™ C tubes (Miltenyi Biotec, PN: 130-093-237) containing 2.5 mL Dulbecco’s modified Eagle media (DMEM), 100 µL proprietary Enzyme D, 50 µL Enzyme R and 12.5 µL Enzyme A from Multi Tissue Dissociation Kit 1 (Miltenyi Biotec, PN: 130-110-201). Tubes were inverted and placed onto gentleMACS™ OctoDissociator with sleeve (Miltenyi Biotec, PN: 130-096-427) and a fibroblast dissociation protocol was run for 1 hour. Enzymatic activity was inactivated with DMEM supplemented with 10% fetal bovine serum (FBS). Cells were gently resuspended via pipetting and run through MACS® 70 µm SmartStrainer (Miltenyi Biotec, PN: 130-098-462), followed by filtration via Scienceware FlowMi® 40 µM Cell Strainers for p1000 pipettes (Sigma Aldrich, PN: H13680-0040). Cells were pelleted and washed with DMEM with 10% FBS. Cells were resuspended in HBSS with 0.04% bovine serum albumin (BSA) prior to library preparation.
Isolated cells were counted on a hemocytometer and prepared using the 10x Genomics Chromium Single-Cell Gene Expression kit (V2, PN: 120267) according to manufacturer’s recommendations. Cells were encapsulated into droplets via gel bead-in emulsion (GEM) method for barcoding using the 10x Genomics Chromium controller (PN: 1000202). GEMs were incubated for cDNA synthesis, followed by amplification and library construction, according to manufacturer’s instructions.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
Illumina MiSeq |
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| Description |
10x Genomics
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| Data processing |
Demultiplexing, barcode processing, feature counting and aggregation was performed using Cell Ranger pipeline (10x Genomics, v3.1.2)
Assembly: Green anole (A. carolinensis) genome FASTA and GTF files (v2.105) were used to generate a reference genome for indexing by Cell Ranger mkref function (v3.1.2, 10x Genomics). The reference genome FASTA and GTF files were sourced from the Ensembl database for green anole, AnoCar2.0v2 (GCA_000090745.2), ensembl release 105. (http://dec2021.archive.ensembl.org/Anolis_carolinensis/Info/Index).
Supplementary files format and content: Tab-separated values files and matrices
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| Submission date |
Jun 13, 2023 |
| Last update date |
Jun 20, 2023 |
| Contact name |
Thomas P Lozito |
| E-mail(s) |
lozito@usc.edu
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| Organization name |
University of Southern California
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| Department |
Orthopaedic Surgery
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| Street address |
1450 Biggy St NRT4513
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90033 |
| Country |
USA |
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| Platform ID |
GPL23678 |
| Series (1) |
| GSE234876 |
Gene expression profile at single-cell level of regenerating lizard (Anolis carolinensis) tail |
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| Relations |
| BioSample |
SAMN35726174 |
| SRA |
SRX20672644 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7476135_D21Obarcodes.tsv.gz |
22.5 Kb |
(ftp)(http) |
TSV |
| GSM7476135_D21Ofeatures.tsv.gz |
177.2 Kb |
(ftp)(http) |
TSV |
| GSM7476135_D21Omatrix.mtx.gz |
12.6 Mb |
(ftp)(http) |
MTX |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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