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| Status |
Public on Dec 12, 2017 |
| Title |
1772092305_B04 |
| Sample type |
SRA |
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| Source name |
dentate gyrus, CD-1, postnatal day 23
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| Organism |
Mus musculus |
| Characteristics |
strain/background: CD-1
genotype/variation: wild type
hgfap-gfp mrna detected (0=no, 1=yes): 0
Sex: Not determined
postnatal day: 23
tissue: dentate gyrus
cell cluster: Granule-mature
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| Growth protocol |
Male and female wild type CD-1 mice (Charles River), as well as hGFAP-GFP reporter mice (Zhuo et al., Developmental Biology, 187(1), p36, 1997), between postnatal days 8 and 68 were used.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were sacrificed by an overdose of Isoflurane or Ketamin/Xylazine, followed by perfusion through the left ventricle with artificial cerebrospinal fluid, equilibrated in 95%O2/5%CO2 on ice or 4°C. The dentate gyrus was then microdissected from 300 μ m vibratome brain sections. Single-cell suspensions were prepared using the Papain kit (Worthington) with 25-35min enzymatic digestion followed by manual trituration using a BSA-coated p1000 pipette or fire-polished Pasteur pipettes. For hGFAP-GFP mice, GFP-positive cells were sorted on a BD FACSAria II into oxygenated aCSF at 4°C. Cells were loaded at 500-800 cells/μ l at 4° on the C1 Fluidigm medium-sized chip.
Lysis mix, RT mix and PCR mix (described in Islam et al., Nat Methods. 2014 Feb;11(2):163-6) were added to the chip. The plate was placed in the Fluidigm C1 instrument and the 'mRNA Seq: RT + Amp (1772x/1773x)' script was executed, and included lysis, reverse transcription and 21 cycles of PCR. The amplified cDNA was harvested in a total of 13 μL C1 Harvesting Reagent. Amplified cDNA was simultaneously fragmented and barcoded by tagmentation using Tn5 DNA transposase to transfer adaptors to the target DNA as described (Ibid.). 100 µl Dynabeads MyOne Streptavidin C1 beads (Invitrogen) were washed in 2x BWT (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 M NaCl, 0.02% Tween-20) and resuspended in 2 ml 2x BWT. Twenty microliters of beads were added to each well and incubated at room temperature for 15 min. All fractions were pooled, the beads were immobilized and the supernatant removed. The beads were resuspended in 100 μL TNT (20 mM Tris, pH 7.5, 50 mM NaCl, 0.02% Tween), washed in 100 μL Qiagen QIAquick PB, and twice in 100 μL TNT. The beads were resuspended in 100 μL restriction mix (1x NEB CutSmart, 0.4 U/μL PvuI-HF enzyme), designed to cleave 3' fragments carrying a PvuI recognition site. The mix was incubated for 1 h at 37 °C, then washed three times in TNT. Finally, to elute DNA, beads were resuspend in 30 µL ddH2O and incubated 10 minutes at 70°C. Beads were then immediately bound to magnet and the supernatant was collected. To remove short fragments, Ampure beads (Beckman Coulter) were used at 1.8x volume and eluted in 30 µL.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
Processed data file: C1_expression_data.tab.gz
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| Data processing |
Read processing was performed as described (Islam et al., Nat Methods. 2014 Feb;11(2):163-6), except that we removed any RNA molecule (i.e., Unique Molecular Identifier) supported only by a single read ("singleton molecules"). This removed a large number of false positive molecules, artefacts that can arise by sequencing error, PCR-induced mutations or translocations and cross-contamination.
The first 6 bases of each read represent the random Unique Molecular Identifier used for molecule counting. After follows three or more Gs, stemming from the template switching at the mRNA 5' end during first strand cDNA synthesis.
Genome_build: UCSC mm10 (GRCm38)
Supplementary_files_format_and_content: C1_expression_data.tab: Tab-delimited text file of total number of detected mRNA molecules from each gene in each cell.
Supplementary_files_format_and_content: datasetB_cellid_to_sampleid.txt: Tab-delimited text file shows the mapping from the sample IDs used in the referenced publication to the cell IDs in this dataset.
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| Submission date |
Mar 06, 2017 |
| Last update date |
Jan 21, 2020 |
| Contact name |
Sten Linnarsson |
| Organization name |
Karolinska Institutet
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| Department |
Medical Biochemistry and Biophysics
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| Lab |
Molecular Neurobiology
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| Street address |
Scheeles väg 1
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| City |
Stockholm |
| ZIP/Postal code |
171 65 |
| Country |
Sweden |
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| Platform ID |
GPL13112 |
| Series (2) |
| GSE95752 |
Transcriptome analysis of single cells from the mouse dentate gyrus [C1] |
| GSE95753 |
Transcriptome analysis of single cells from the mouse dentate gyrus |
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| Relations |
| BioSample |
SAMN06482309 |
| SRA |
SRX2615301 |
| 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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