GSM3185263
GSM3185263: single_cell_RNAseq [s_dot_ogp67]; Mus musculus; RNA-Seq
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. SVZs from 2 mice of 2 or 4 mice of 22 months of age were pulled per replicate. Single cell suspension was obtained using the Neural Tissue Dissociation kit with Trypsin (or Papain when staining for CD31+ and CDd11b+ cells) and Gentle MACS dissociator (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: O4 APC-Cy7 (Miltenyi, 1:50), CD45 APC-Cy7 (BD, 1:200), GLAST(ACSA-1)-PE (Miltenyi, 1:20), Prom-APC, PSA-NCAM PE-Cy7 Miltenyi, 1:75), CD31-PE (BD, 1:100) and CD11b-APC (BD, 1:100). 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 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. For single cell RNA-seq, we sorted for CD45-O4-CD133+GLAST+, cells. Single NSCs were handpicked and libraries according to Smartseq2 were prepared. For Bulk sequencing we sorted 1000 events of different populations based on the following marker combinations: Endothelial CD45-CD31+, Microglia CD45+Cd11b+, NSCs CD45-CD133+GLAST+, Neuroblasts CD45-GLAST-PSA-NCAM+. 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 or 3000.
GEO
GSM3185263
GEO Accession
GSM3185263