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| Status |
Public on Feb 03, 2018 |
| Title |
P5_2 |
| Sample type |
SRA |
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| Source name |
Lateral geniculte nucleus
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| Organism |
Mus musculus |
| Characteristics |
tissue: Lateral geniculte nucleus
strain: C57BL/6
age: Post-natal day 5
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| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were euthanized on postnatal days 5, 10, 16, and 21. Each time point includes data from eight mice total processed as four independent samples of two mice each. After isoflurane anesthetization, mice were decapitated and the brain was isolated. Mouse brains were dissected and 300 _m coronal sections were made on a Leica VT1000S vibratome. The dorsal LGN were then microdissected in ice cold PBS after visual identification using a Nikon SMZ-10A brightfield dissection microscope. LGNs from four animals were pooled to create one replicate per time point. LGN tissue was transferred to dissociation media: HBSS (Life Technologies), 10mM HEPES (Sigma), 172mg/l kynurenic acid (Sigma), 0.86 g/l MgCl2o6H2O (Sigma), 6.3 g/l D-glucose (Sigma). This solution was saturated with 95% oxygen and 5% CO2 and was pH adjusted to 7.35 prior to use. Papain (20U/ml, Worthington), Pronase from Streptomyces griseus (1 mg/ml, Sigma), proteinase XXIII from Aspergillus melleus (3 mg/ml, Sigma) and DNAse (2mg/ml, Worthington) were added to the dissociation media. Dissociation was carried out at 37¡ C for 1h. The samples were then triturated, filtered, and spun at 300 x g for five minutes. The pellet was resuspended in trypsin inhibitor solution (dissociation media with 1% BSA and 1% ovomucoid). Following gradient centrifugation, the cells were washed in dissociation media containing 0.04% BSA and resuspended in dissociation media containing 0.04% BSA and 15% Optiprep (Sigma).
Individual cells were captured and barcoded using the inDrops platform as previously described. Briefly, single-cell suspensions were fed into a microfluidic device that packaged the cells with barcoded hydrogel microspheres and reverse transcriptase/lysis reagents. After cell encapsulation, primers were photo-released by UV exposure. Two libraries of approximately 3000 cells each were collected for each sample. Indexed libraries were pooled and sequenced on a Nextseq 500 (Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
single cell isolation by Indrops
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| Data processing |
Transcripts were processed according to the pipeline in Macosko et al. (2015). Briefly, this pipeline was used to build a custom transcriptome from Ensembl GRCm38 genome and GRCm38.84 annotation using Bowtie 1.1.1, after filtering the annoatation gtf file (gencode.v17.annotation.gtf filtered for feature_type=îgeneî, gene_type="protein_coding" and gene_status="KNOWN"). Read quality control and mapping against this transcriptome was performed. Unique molecular identifiers (UMIs) were used to link sequence reads back to individual captured molecules. All steps of the pipeline were run using default parameters unless explicitly stated.
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| Submission date |
Jan 30, 2018 |
| Last update date |
Feb 03, 2018 |
| Contact name |
Brian Kalish |
| E-mail(s) |
brian.kalish@sickkids.ca
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| Phone |
6305329007
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| Organization name |
Harvard Medical School
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| Department |
Neurobiology
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| Lab |
Greenberg Lab
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| Street address |
220 Longwood Ave
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE108761 |
Single-cell transcriptomics of the developing lateral geniculate nucleus reveals insights into circuit assembly and refinement |
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| Relations |
| BioSample |
SAMN08457969 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2971229_P5_2.counts.tsv.gz |
21.0 Mb |
(ftp)(http) |
TSV |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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