Illumina HiSeq 2000 paired end sequencing; GSM1656767: I1; Mus musculus; RNA-Seq

Heterogeneous pools of adult neural stem cells (NSCs) contribute to brain maintenance and regeneration after injury. The balance of NSC activation and quiescence, as well as the induction of lineage-specific transcription factors, may contribute to diversity of neuronal and glial fates. To identify molecular hallmarks governing these characteristics, we performed single-cell sequencing of an unbiased pool of adult subventricular zone NSCs. This analysis identified a discrete, dormant NSC subpopulation that already expresses distinct combinations of lineage-specific transcription factors during homeostasis. Dormant NSCs enter a primed-quiescent state before activation, which is accompanied by downregulation of glycolytic metabolism, Notch, and BMP signaling and a concomitant upregulation of lineage-specific transcription factors and protein synthesis. In response to brain ischemia, interferon gamma signaling induces dormant NSC subpopulations to enter the primed-quiescent state. This study unveils general principles underlying NSC activation and lineage priming and opens potential avenues for regenerative medicine in the brain. Overall design: Single cell RNAseq of cells isolated from their in vivo niche in the subventricular zone, Striatum and Cortex during homeostasis as well as following ischemic injury. In total 272 single cells. (<WT>: homeostasis samples; <Ischemic_injured> and <Ischemic_injured_and_Interferon_gamma_knockout>: samples following ischemic injuried).

Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury

Submitted by Gene Expression Omnibus on 02-AUG-2015

Animals were sacrificed by cervical dislocation and their brains immediately placed on ice. The lateral SVZ was microdissected as a whole mount as previously described 2. Pooled SVZ tissue (1-5 mice) was digested with trypsin and DNase according to the Neural Tissue Dissociation kit in a Gentle MACS dissociatior (Miltenyi) to maximize cell viability and recovery. Cells were stained for 20 minutes on ice in DPBS (without Ca and Mg) supplemented with 10% fetal calf serum. Combinations of the following antibodies were used: APC and PE anti-GLAST (ACSA-1, Miltenyi, 1/40), APC anti O4 (Miltenyi, 1:40), PE-Vio770 anti PSA-NCAM (Miltenyi, 1/50), APC and PerCP-eFluor710 anti-Prominin-1 (eBioscience, 1/75), Alexa488::EGF (Life Technologies, 1/100), APC-Cy7 anti-CD45 (BD, 1/200). Sytox Blue (Life Technologies, 1/1000) was added to the cell suspension prior to sorting. Stained cell suspensions were sorted in a FACSAriaII (BD) using a 100um nozzle at 20psi. Cells were collected in ice-cold Neurobasal medium supplemented with L-Glutamine, B27 and Penicillin/Streptomycin at a density of approx. 1cell/ul. Immediately after sorting cells were manually picked with a thin gel-loading micropipette under continuous visual inspection in a Zeiss microscope. To ensure that only one cell was picked, we transferred sequentially 1ul of the cell suspension to the picking plate where a single cell was immediately picked in 0.3ul with another pipette before the addition of a new cell to the plate. Cells were directly transferred to ice-cold lysis buffer (0.2%Triton X-100 containing RNAse inhibitors, Clontech) and frozen at -80ºC until library preparation. Cells labeled as PSA, were sorted according to CD45negPSA-NCAMposGLASTneg; all the other cells were CD45negPSA-NCAMnegGLASTposPROM1pos. RNA-seq libraries were prepared using Smart-seq2 technology as previously described. Briefly, lysed cells were thawed and subject to reverse transcription using an oligo(dT) primer and a locked nucleic acid (LNA)-containing template-switching oligonucleotide (Exiqon). In a randomly selected set of samples we included ERCC Spike-Ins (Ambion) at a 1:1000000 dilution. Full-length cDNAs were amplified by 20 cycles of PCR using KAPA HiFi DNA polymerase (KAPA biosystems). Amplified cDNAs for every single cell were quantified and run on a High Sensitivity Bioanalyzer chip (Agilent). Samples containing no material or in which the cDNA appeared degraded were excluded (approx. 5-10%) from further processing. cDNAs were then converted into libraries for Illumina sequencing according to the Nextera XT Sample Preparation (Illumina) protocol with minor modifications. Specifically, we extended the tagmentation step to 8 minutes and performed a double cleanup with 0.8X AMPure XP SPRI beads (Agencourt) after PCR amplification for 9 cycles. To control for technical variability we performed two replicate DNA libraries with cDNA from the same cell. For population studies, approx. 1ng of total RNA from 1000 cells was subject to SMARTer Ultra Low RNA kit (Clontech) according to the manufacturer’s instructions. cDNA products were quantified, run in a Bioanalyzer and subject to Illumina library preparation according to NEBNext  Ultra DNA Library Preparation Kit (NEB). Libraries were quantified, pooled at equal concentrations and the multiplex was cleaned up again with 1X AMPure beads and measured in a Bioanalyzer prior to 101bp paired-end sequencing in an Illumina HiSeq 2000.

Single-Cell Transcriptomics Reveals a Population of Dormant Neural Stem Cells that Become Activated upon Brain Injury

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