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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Aug 22, 2018 |
| Title |
1772078033_A04 |
| Sample type |
SRA |
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| Source name |
striatum
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| Organism |
Mus musculus |
| Characteristics |
tissue: brain
brain region: dorsolateral striatum
strain: Lhx6icreRFP
cell type: ENDO
gfp fluorescence: 0
rfp fluorescence: 0
age (days): 22
Sex: male
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| Growth protocol |
Two mouse transgenic lines on CD1 background were used in order to enrich the interneuron populations. The Lhx6-Cre RFP, a BAC transgenic mouse that express Cre under transcriptional control of Lim homeobox 6, together with the expression of the tandem-dimer red fluorescent protein (tdRFP) (ubiquitously expressed under the ROSA26 locus. And the BAC transgenic mouse 5HT3a-EGFP, where EGFP is expressed under the control of the Htr3a promoter. The mice were between postnatal day 22-28 and comprised both sexes.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Mice were deeply anesthetized, and the brain was quickly dissected and sectioned on a vibratome (VT1200 S, Leica) in 300 micrometer thick slices. The dorso-lateral striatum (bregma, AP: 1.42 to -0.58 mm) was dissected from each slice, and the tissue was dissociated using the Papain dissociation system (Worthington). The cell suspension obtained was filtered with 20 micrometer filter (Partec). RFP+ or EGFP+ cells were immediately FACS sorted using a BD FACSAria III Cell Sorter B5/R3/V3 system. 7 microliter of both the fluorescent and non-fluorescent populations at 600-1000 cells/microliter had 4 microliter of C1 Cell Suspension Reagent added, and loaded on a C1 Single-Cell AutoPrep IFC microfluidic chip designed for 10-17-micrometer cells (Fluidigm Inc.).
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 2× BWT (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 2 M NaCl, 0.02% Tween-20) and resuspended in 2 ml 2× 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 (1× 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 1.8× 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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| 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 sythesis.
Genome_build: UCSC mm10
Supplementary_files_format_and_content: text table of total number of detected mRNA molecules from each gene in each cell
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| Submission date |
Apr 06, 2017 |
| Last update date |
May 15, 2019 |
| 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) |
| GSE97478 |
Single cell RNA sequencing of interneurons of the mouse dorsolateral striatum I |
| GSE106708 |
Single cell RNA sequencing of interneurons of the mouse dorsolateral striatum |
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| Relations |
| BioSample |
SAMN06696725 |
| SRA |
SRX2738135 |
| 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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