title: C1-1772099-009-G04


title: C1-1772099-009-G10

status: Public on Jun 10 2016

type: SRA

city: Stockholm

first: Sten

last: Linnarsson

address: Scheeles väg 1

department: Medical Biochemistry and Biophysics

country: Sweden

institute: Karolinska Institutet

library_source: transcriptomic

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: Tab-delimited table of total number of detected mRNA molecules from each gene in each cell

molecule: polyA RNA

organism: Mus musculus

source_name: SN-VTA

growth_protocol: Wild-type (CD1 or C57BL6) and transgenic mice (5Pdgfratm11(EGFP) Sor/J, Lhx6 Cre RFP and PDGFR-GFP) between post-natal day 21 and 31 of both sexes were used. In the PDGFRa-GFP knock-in mice, H2B-eGFP fusion gene is expressed under the promoter of the oligodendrocyte precursor marker PDGFRa. The Lhx6–Cre transgenic mouse is a BAC transgenic mouse that expresses Cre under transcriptional control of Lhx6. This mouse line was crossed with the Cre-reporter line where tandem-dimer red fluorescent protein (tdRFP) is ubiquitously expressed under the ROSA26 locus. In the BAC transgenic mouse 5HT3aEGFP, EGFP is expressed under control of the Htr3a promoter.

extract_protocol:
Selected CNS regions were dissected in 300µm thick vibratome sections and cut out with scalpel. Tissue was then dissociated into a single cell suspension (described in Zeisel & Munoz-Manchado, Science 2015 Mar 6;347(6226):1138). For the spinal cord, an extra gradient step was used after the strainer and final centrifugation step. The pellet was ressuspended in 2ml Neurobasal-A medium including supplements and transferred onto an Optiprep Density Gradient, which consisted of 3 phases (Bottom phase: 160µl Optiprep + 340µl Neurobasal-A; middle phase: 220µl Optiprep + 1780µl Neurobasal-A; top phase: 75µl Optiprep + 925µl Neurobasal-A). The gradient was then centrifuged at 300g for 10min at 8°C and cells were located between the bottom and the middle phase. Cell suspension in a concentration of 600-1000 cells/μL was used. C1 Suspension Reagent was added in a ratio of 4 μL to every 7 μL cell suspension. 11 μL of the cell suspension mix was loaded on a C1 Single-Cell AutoPrep IFC  microfluidic chip designed for 10- to 17-μm cells, and the chip was then processed on a Fluidigm C1 instrument using the 'mRNA Seq: Cell Load (1772x/1773x)' script (30 min at 4°C).
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.

treatment_protocol: In the stress treatment mice, acute stress was induced injection of 4% PFA into the left paw.

tag: strain

tag: age

tag: Sex

value: pooled male and female

tag: treatment

value: No

tag: inferred cell type


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source_name: dorsal horn


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source_name: hypothalamus

value: M

value: 6hr post acute stress


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source_name: cortex S1


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