title: GSM1684700: Single_cell_RNASeq [ctx17]; Mus musculus; RNA-Seq
library_construction_protocol: 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. TAP cells were isolated from the SVZ based on EGFpos GLAST/CD45/PSA-NCAM/O4neg. Str refers to GLASTpos CD45/O4neg cells from the striatum (SVZ was excluded). Str refers to GLASTpos CD45/O4neg from the somatosensory cortex. 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.
attributes.tag: GEO Accession
attributes.value: GSM1684700