|
| Status |
Public on Apr 19, 2019 |
| Title |
JH17_Cerebral_cortex_hypoxia_scRNAseq |
| Sample type |
SRA |
| |
|
| Source name |
adult dissociated cerebral cortex, 7-day hypoxia
|
| Organism |
Mus musculus |
| Characteristics |
genotype: WT
strain: C57BL/6J
age: 2 month
Sex: Male
tissue: cerebral cortex
treatment: Mouse was exposed to normoxic conditions
|
| Growth protocol |
Adult mice were housed in our animal facility with 12 h light/dark cycles and food ab libitum. Animals were used for analysis in accordance with protocols approved by the Institutional Animal Care and Use Committee
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Dissociated single cells were processed through the GemCode Single Cell Platform using the GemCode Gel Bead, Chip, and Library Kits (10X Genomics, Pleasanton) as per the manufacturer’s protocol. In brief, single cells were sorted into 0.4% BSA–DPBS. Approximately 14,000 single retinal cells were added to a channel. The cells were then partitioned into Gel Beads in Emulsion in the 10x Genomics Chromium Controller, where cell lysis
Cell lysis was followed by barcoded oligo-DT priming and reverse transcription of poly-adenylated RNA, amplification, shearing and 5′ adaptor and sample index attachment.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Dissociated cerebral cortical cells
|
| Data processing |
scRNA-seq data were pre-processed using the Cell Ranger pipeline (v2.1.0; 10x Genomics) with default settings. The normalized gene x cell matrix was used as input for the MonocleR/Bioconductor package (Trapnell et al., 2014). Log-normalized expression estimates (with a pseudocount of 1) were used as input for a modified dpFeature analysis. For preliminary cell clustering, PCA was performed on the high variance genes, and appropriate components were visualized in two dimensions using UMAP. Initial clusters were identified from the 2-D embedding using k-means clustering. Cell type identity was determined by cross-referencing clusters with expression of multiple known cell type markers.
RiboTag, whole retina, and whole brain RNA-seq data were pseudoaligned to the mouse genome (mm10) using the Kallisto software (Bray et al., 2016). Aligned counts were obtained for differential gene expression analysis using EBseq (Leng et al., 2013) and posterior probability ³0.95 that a gene/transcript is differentially expressed was considered statistically significant.
Genome_build: mm10
|
| |
|
| Submission date |
Jan 27, 2019 |
| Last update date |
Apr 19, 2019 |
| Contact name |
Jacob S Heng |
| E-mail(s) |
jacob.heng@outlook.com
|
| Organization name |
Johns Hopkins University School of Medicine
|
| Department |
Molecular Biology and Genetics
|
| Lab |
Nathans Lab
|
| Street address |
725 North Wolfe Street
|
| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21210 |
| Country |
USA |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE125708 |
Hypoxia tolerance in the Norrin-deficient retina and the chronically hypoxic brain stuied at single-cell resolution |
|
| Relations |
| BioSample |
SAMN10826877 |
| SRA |
SRX5296675 |
