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Sample GSM4623794 Query DataSets for GSM4623794
Status Public on Apr 30, 2021
Title Epithelial progenitors E18.5
Sample type SRA
 
Source name Trachea epithelial progenitors
Organism Mus musculus
Characteristics strain: C57BL/6
developmental stage: E18.5
Treatment protocol None
Growth protocol None
Extracted molecule polyA RNA
Extraction protocol To prepare single-cell suspensions of epithelial cells in 7 time points (E12.5, E13.5, E14.5, E16.5, E18.5, and 2months), the epithelial sheet was peeled off from mesenchymal tissue in developing trachea with a tungsten needle after the incubation with 175U/ml collagenase typeⅠ (Worthington Biochemical Corporation, CLS1) at 37℃ for 6 - 120 min. Single-cell suspensions were made through the incubation with Trypsin-EDTA (0.25%) (Thermo Fisher Scientific, 25200056) at 37℃ for 15 min, then loaded onto Chromium Single Cell A Chips (10X Genomics, PN-1000009) for the Chromium Single Cell 3′ Library v2 (10X Genomics, PN-120233) according to the manufacturer’s recommendations (10X Genomics)
Signle-cell suspensions were loaded onto Chromium Single Cell A Chips (10X Genomics, PN-1000009) for the Chromium Single Cell 3′ Library v2 (10X Genomics, PN-120233) according to the manufacturer’s recommendations (10X Genomics). Briefly, single-cell gel bead-in-emulsions (GEMs) were generated from loaded cell suspensions by a Chromium Controller instrument (10X Genomics). After performing GEM-reverse transcriptions (GEM-RTs), GEMs were harvested and the cDNAs were amplified and cleaned up with SPRIselect Reagent Kit (Beckman Coulter, B23318). Indexed sequencing libraries were constructed using Chromium Single Cell 3′ Library v2 (10X Genomics, PN-120233) for enzymatic fragmentation, end-repair, A-tailing, adaptor ligation, ligation cleanup, sample index PCR, and PCR cleanup. Libraries were sequenced on a HiSeq1500 (Illumina) to obtain a sequencing depth of around 50,000 reads per cells.
single cell RNA-seq using Chromium system (PolyA+ RNA)
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 1500
 
Data processing Basecalls performed using Illumina RTA 1.18.64 and the fastq files were generated by Cellranger v2.2.0 and bcl2fastq 2.19.0.316
signle cell RNA-seq reads were aligned to the mm10 genome reference using Cellranger v2.2.0 with default parameters
Seurat v2.3.4: the cells meeting any of the following criteria were omitted from further analyses for the quality control; <1,000 or >5,000 UMIs, > 7.5% of reads mapping to mitochondria genes, or EpCAM negative cells. For clustering, principal-component analysis was performed for dimension reduction. Top 15 principal components (PCs) were selected by using a permutation-based test implemented in Seurat and passed to t-Distributed Stochastic Neighbor Embedding (tSNE) for clustering visualization. To maintain a standard procedure for clustering, a value of 0.8 for the resolution was used
Genome_build: mm10
Supplementary_files_format_and_content: Filtered gene-barcode matrix of CellRanger output including sparse matrix (MEX), cellular barcodes (TSV), and gene list (TSV). Processed data with Seurat (Robj)
 
Submission date Jun 17, 2020
Last update date Apr 30, 2021
Contact name Hirofumi Kiyokawa
E-mail(s) h.kiyokawa@gmail.com
Phone 81783063199
Organization name Riken
Department Center for Biosystems Dynamics Research
Lab Laboratory for lung development and regenration
Street address 2-2-3 Minatojima-minamimachi, Chuo-ku
City Kobe
State/province Hyogo
ZIP/Postal code 6500047
Country Japan
 
Platform ID GPL18480
Series (1)
GSE152692 Comprehensive single cell RNA-seq datasets of mice trachea epithelial progenitors in 6 time points from E12.5 to E18.5 and mature epithelial cells in 2-month mice
Relations
BioSample SAMN15300198
SRA SRX8567945

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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