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Sample GSM5945756 Query DataSets for GSM5945756
Status Public on Sep 02, 2022
Title E4_2
Sample type SRA
Source name Telencephalon
Organism Pleurodeles waltl
Characteristics animal: A4
brain region: Telencephalon
Treatment protocol No treatment
Growth protocol Animal grown in lab to adulthood
Extracted molecule total RNA
Extraction protocol For brain dissociation, animals were deeply anesthetized by submersion in 0.2% MS-222. The brain was perfused transcardially with ice-cold oxygenated Amphibian Ringer’s solution (96 mM NaCl; 20 mM NaHCO3; 2 mM KCl; 10 mM HEPES; 11 mM glucose; 2 mM CaCl2; 0.5 mM MgCl2), and the animal decapitated. The whole brain or telencephalon was dissected out, and embedded in 4% LM Agarose in Ringer. The brain was then sliced coronally on a vibratome into 500 um sections, and cut into 500 um cubes in cold carbogenated Ringer. The tissue pieces were transferred to a 5 mL tube in 2.5 mL Dissociation Buffer (20 U/mL Papain, 200 U/mL DNase, 5 ug/mL Liberase, 1 uM TTX), and incubated at RT for 30 minutes on a rotator. Following enzymatic dissociation, the tissue was mechanically dissociated by trituration with fire polished, silanized glass pipettes of decreasing tip diameter on ice. The supernatant was passed through a 100 um cell strainer, with fresh carbogenated Hibernate A-Ca media added between each subsequent pipette until a uniform suspension was obtained. Hibernate A-Ca was added up to a total volume of 20 mL, and the solution was passed through a 70 um cell strainer. To the bottom of the tube, 5 mL of 4% BSA in Hibernate A-Ca - phenol red was added. To filter out cell debris, the cell suspension was centrifuged through the density gradient at 300xg (4°C) for 5 min. The supernatant was removed, and the pellet resuspended in 50-100 uL Hibernate A-Ca - Mg. The cell concentration was determined by counting on a Fuchs- Rosenthal chamber with trypan blue, and diluted to 1000 cells/uL in Hibernate A- Ca - Mg
The cells were loaded into a 10x Chromium Chip G with a targeted cell recovery of 5000-8000 cells for GEM Generation and cell barcoding. Single cell RNA-seq libraries were prepared with 10x Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.0 or v3.1 (Dual Index)
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina NovaSeq 6000
Data processing Reads were assigned to the combined P.waltl transcriptome using Alevin (Salmon v1.6.0). Library type was set to automatic and keepCBFraction was set to 1. All other parameters were set as default.
Count matrices were obtained after alignment with Alevin (Salmon v1.6.0) and knee plots were generated for each library. Low UMI counts were filtered out and a single Seurat object containing all libraries was created using Seurat 4.0.2.
Assembly: p.waltl_transcriptome. FASTA and t2g files are available on the SuperSeries record.
Supplementary files format and content: .rds of count_matrix
Submission date Mar 10, 2022
Last update date Sep 02, 2022
Contact name Maria ANTONIETTA Tosches
Organization name Columbia University
Street address 1212 Amsterdam Ave
City New York
ZIP/Postal code 10027
Country USA
Platform ID GPL32009
Series (2)
GSE198363 Cell type profiling in salamanders identifies innovations in vertebrate forebrain evolution [E4]
GSE198367 Cell type profiling in salamanders identifies innovations in vertebrate forebrain evolution
BioSample SAMN26565315
SRA SRX14430756

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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