|
| Status |
Public on Aug 12, 2019 |
| Title |
sc-N2_2_pooling_after |
| Sample type |
SRA |
| |
|
| Source name |
sc-N2_2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Craig Venter-B (CVB) hiPSC subline (hiPSC2)
cell type: hiPSC-derived single-cell neuron
|
| Treatment protocol |
No particular treatment was applied after differentiation.
|
| Growth protocol |
hiPSCs were seeded onto PA6 cells in PA differentiation medium for 12 days. Between days 12-14, when rosettes were visible, after dissociation, were FACS-sorted based on the CD184+/CD271-/CD44-/CD24+ cell-surface signature. Sorted NPCs were cultured on 20µg/mL poly-L-ornithine and 5µg/mL laminin coated plates in NPC medium until confluency was reached. To generate neurons, NPCs grown in 10 cm-plates at confluency were switched to NPC medium without FGF (day 1), and cultured afterwards for 3 weeks. Around day 21, neurons were sorted based on the CD184-/CD44-/CD24+ signature. For our experiments 1x10^6 CD24+ sorted neurons were plated on 20µg/mL poly-L-ornithine and 5µg/mL laminin coated 24-well plates in NPC media supplemented with 0.5mM dbcAMP from Sigma, and 20ng/µL BDNF and 20 ng/µL GDNF. After 10 days recovery, neuronal cultures were sorted for single cell isolation and individual cells were processed for scRNA-seq.
|
| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted and processed following the SMART-seq protocol from Takada and NEXTERA from Illumina, following in both cases the Takada's protocol that allows full-length coverage of gene expression at the level of single cell.
Samples were processed using the SMART-seq protocol from Takada and NEXTERA from Illumina, following in both cases the Takada's protocol that allows full-length coverage of gene expression at the level of single cell.RNA quality. DNA quality and size was examined for every individual cells using Bioanalyzer (Agilent Technologies). Only samples with detectable DNA and size in accord with not prior RNA degradation were selected for sequencing. Libraries were pooled before cleaning up and final DNA concentration measuring (these samples represent "pooling_before," or [a] files; while the same libraries were pooled after. cleaning and final DNA concentration measuring (these samples represent "pooling_after," or [b] files; in short, therefore, each single cell library was sequenced twice, in onec case the exact concentration of each library in the pool was not adjusted individually, while in the other case it was adjusted before pooling; these two options are suggested in the Takada's protocol and we aimed to test both).
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
Induced differentiation from hiPSCs
2a_S2_L008
|
| Data processing |
Reads were aligned to the hg18 human genome with bowtie2 (–very-sensitive option) and tophat. One mismatch was allowed.
Artifacts derived from clonal amplification were circumvented by considering maximal three tags from each unique genomic position as determined from the mapping data
Read counts were calculated with HOMER 3.9 considering only exonic regions of human RefSeq genes
The bedGraph and bigwig files were generated by using Homer v3.9, which the total tags are normalized to 1.00e+07.
Genome_build: hg18
Supplementary_files_format_and_content: bedGraph
|
| |
|
| Submission date |
Aug 09, 2019 |
| Last update date |
Aug 14, 2019 |
| Contact name |
Daria Merkurjev |
| E-mail(s) |
[email protected]
|
| Phone |
858-534-5858
|
| Organization name |
UCSD
|
| Department |
Medicine
|
| Lab |
Michael G. Rosenfeld Laboratory
|
| Street address |
9500 Gilman Drive, Mail Code 0648
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
| |
|
| Platform ID |
GPL20301 |
| Series (2) |
| GSE106872 |
Analysis of the Clustered Protocadherin (cPcdh) Locus in Human Pluripotent Stem and Derived Cells |
| GSE135634 |
Analysis of the Clustered Protocadherin (cPcdh) Locus in Human Pluripotent Stem and Derived Cells [scRNA-seq] |
|
| Relations |
| BioSample |
SAMN12542826 |
| SRA |
SRX6687135 |
