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| Status |
Public on Dec 31, 2019 |
| Title |
YN14_056_PanTro_NeuN |
| Sample type |
SRA |
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| Source name |
brain
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| Organism |
Pan troglodytes |
| Characteristics |
tissue: brain
nuclei: NeuN
tissue preparation: Frozen Brain Tissue
region: Brodmann Area 46
sample id: YN14.056
gender: F
age: 23
humanized age: 45
hemisphere: L
pmi: 0.9
rin: 6.6
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| Extracted molecule |
total RNA |
| Extraction protocol |
nuclear extraction protocol: Tissue isolated from Brodmann Area 46 from 27 healthy humans, 11 healthy chimpanzee and 15 healthy rhesus macaque.700 mg of frozen postmortem brain was homogenized with lysis buffer using a dounce homogenizer. Brain lysate was placed on a sucrose solution to create a concentration gradient. Ultracentrifuge was used to separate nuclei and cytoplasmic fraction.
FANS protocol: Nuclei were resuspended in PBS plus RNase inhibitors and incubated with mouse Alexa488 conjugated anti-NeuN (1:200) (#MAB377X, Millipore, Billerica, MA) and rabbit Alexa555 conjugated anti-OLIG2 (1:75) (#AB9610-AF555, Millipore) antibodies with 0.5% BSA. Immuno-labeled nuclei were collected as NeuN-positive or OLIG2-positive populations by fluorescence-activated cell sorting (FACS).
total RNA extraction protocol: Total RNA were purified from each nuclei population using a ZR-Duet DNA/RNA MiniPrep (Plus) kit according to the manufacturer's instruction. 200 ng total RNA from each sample was treated for ribosomal RNA (rRNA) removal using the Low Input RiboMinus Eukaryote System v2 (#A15027, ThermoFisher) according to the manufacturer's instruction.
Total RNAs with an average RIN value of 7.5±0.16 were used for RNA-seq library preparation. 50 ng of total RNA after rRNA removal was subjected to fragmentation, first and second strand syntheses, and clean up by EpiNext beads (#P1063, EpiGentek, Farmingdale, NY). Second strand cDNA was adenylated, ligated and cleaned up twice by EpiNext beads. cDNA libraries were amplified by PCR, and cleaned up twice by EpiNext beads. cDNA library quality was quantified by a 2100 Bioanalyzer using an Agilent High Sensitivity DNA Kit (#5067-4626, Agilent, Santa Clara, CA). Barcoded libraries were pooled and underwent 75 bp single-end sequencing on an Illumina NextSeq 500.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Protocol 1
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| Data processing |
Single-end reads were aligned with STAR 2.5.2b using options "“--outFilterMultimapNmax 10 --alignSJoverhangMin 10 --alignSJDBoverhangMin 1 --outFilterMismatchNmax 3 --twopassMode Basic" to the reference sequence (hg19, PanTro5, RheMac8 from UCSC)
Crossmap and Liftover tools were used to translate non-human priamte coordinates into human coordinates. Human reference was used for gene annotation and counting (gencode v19). Uniquely mapped reads were quantified using HTSeq python suite using high-scored orthologs' protein coding genes by exons.
Genome_build: Human hg19, Chimpanzee PanTro5, Rhesus Macaque RheMac8
Supplementary_files_format_and_content: tab separated files with raw counts
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| Submission date |
Dec 17, 2018 |
| Last update date |
Dec 31, 2019 |
| Contact name |
Genevieve Konopka |
| E-mail(s) |
gena@alum.mit.edu
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| Organization name |
UT Southwestern Medical Center
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| Department |
Neuroscience
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| Street address |
5323 Harry Hines Blvd.
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| City |
Dallas |
| State/province |
TX |
| ZIP/Postal code |
75390-9111 |
| Country |
USA |
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| Platform ID |
GPL21121 |
| Series (1) |
| GSE123936 |
Accelerated evolution of oligodendrocytes in human brain |
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| Relations |
| BioSample |
SAMN10602399 |
| SRA |
SRX5139634 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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