|
| Status |
Public on Dec 07, 2015 |
| Title |
whOrg5_G2_hOrg65d |
| Sample type |
SRA |
| |
|
| Source name |
iPSC 409B2
|
| Organism |
Homo sapiens |
| Characteristics |
Stage: 65 days
tissue: Dissociated whole cerebral organoid
|
| Treatment protocol |
Whole cerebral organoids and microdisseccted ventricles were dissociated in 2 ml of Accutase (StemPro) containing 0.2 U/µl Dnase I (Roche) for approximately 45 minutes. Dissociated cells were filtered through 40, 30, and 20 μm diameter strainers to create a single cell suspension.
|
| Growth protocol |
Cerebral organoids were generated using the same protocol as described Lancaster et al. Nature Protocols 2014, with the exception that mTESR1 (Stem Cell Technologies) was used during embryoid body formation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were loaded into the Fluidigm C1 microfluidic platform, where single cells were captured. Lysis of single cells, reverse-transcription of mRNA into cDNA as well as preamplification of cDNA occured within the microfluidic device using reagents provided by Fluidigm as well as the SMARTer Ultra Low RNA kit for Illumina (Clontech). External RNA spike-in transcripts (ERCC spike-in Mix, Ambion) were added to all single cell lysis reactions at a dilution of 1:40,000.
Libraries were prepared using Illumina Nextera XT kit per illumina's protocols.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Single cell G2 from human cerebral organoid experiment at 65d
whOrg5_G2_hOrg65d
|
| Data processing |
"freeIbis" was used as an efficient basecaller with calibrated quality scores for Illumina sequencers. (Bioinformatics. 2013 May 1;29(9):1208-9. doi: 10.1093/bioinformatics/btt117. Epub 2013 Mar 6.)
"leeHom" was used to trim adapters and merge Illumina sequencing reads. (Nucleic Acids Res. 2014 Oct;42(18):e141. doi: 10.1093/nar/gku699. Epub 2014 Aug 6.)
"deML" was used for robust demultiplexing of Illumina sequences using a likelihood-based approach. (Bioinformatics. 2015 Mar 1;31(5):770-2. doi: 10.1093/bioinformatics/btu719. Epub 2014 Oct 30.)
Reads were aligned against the human genome (build GRCh38.77 from ENSEMBLE) using TopHat v2.0.14. The genome was indexed using Bowtie v2.2.6
Fragments Per Kilobase of transcript Per Million mapped reads (FPKM) values were computed using cufflinks v2.2.1
Genome_build: GRCh38
Supplementary_files_format_and_content: Master data frame including sample name, gene ID, and FPKM values for each single cell.
|
| |
|
| Submission date |
Nov 30, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Gray Camp |
| E-mail(s) |
graycamp@gmail.com
|
| Organization name |
Max Planck Institute for Evolutionary Anthropology
|
| Department |
Evolutionary Genetics
|
| Street address |
Deutscher Pl. 6
|
| City |
Leipzig |
| ZIP/Postal code |
04103 |
| Country |
Germany |
| |
|
| Platform ID |
GPL16791 |
| Series (1) |
| GSE75140 |
Human cerebral organoids recapitulate gene expression programs of fetal neocortex development. |
|
| Relations |
| Reanalyzed by |
GSE124299 |
| BioSample |
SAMN04304064 |
| SRA |
SRX1456864 |
