title: Illumina HiSeq 2500 paired end sequencing; Activation of a neural stem cell transcriptional program in parenchymal astrocytes
design: Activation of a neural stem cell transcriptional program in parenchymal astrocytes
library_name: Sample 222_p
library_construction_protocol: One mouse brain was used for each time point, for a total of 4 brains. From each brain, a ~1 mm thick coronal slice was isolated, spanning rostrocaudal levels Bregma -0.2 to +1.0 (Fig. S1a-c). From this section, the SVZ was isolated and discarded. The striatum and cortex were then isolated from both hemispheres. Tissue was cut into fine pieces and incubated with papain solution (20 units/ml) containing L-cysteine (1 mM), EDTA (0.5 mM) and DNase (2000 units/ml) (Papain Dissociation System [LK003160], Worthington, Lakewood, NJ, USA), dissolved in EBSS (Thermo Fisher), at 37°C for 30 minutes, with occasional agitation. Then, the tissue was triturated with smaller and smaller pipet tips and filtered through a 70-μm cell strainer (BD, Franklin Lakes, NJ, USA). After a 10-minute centrifugation at 200*g in a swing-bucket rotor at 4°C, supernatant was removed and cells were resuspended in 3 ml sterile-filtered washing buffer (Dulbecco's PBS containing sodium pyruvate, streptomycin sulfate, kanamycin monosulfate, glucose and calcium chloride; Sigma-Aldrich, D4031) containing 0.5% bovine serum albumin (BSA; Sigma-Aldrich, A9418). To remove debris, we then used gradient centrifugation and myelin removal beads in the following way. The cell suspension was carefully layered on top of 4 ml of 90% Percoll (GE Healthcare, Chicago, IL, USA; 17-0891-01, diluted in 1.5 M sterile-filtered NaCl) in a 15-ml Falcon tube and centrifuged at 200*g for 15 minutes in a swing-bucket rotor at 4°C. After removing the small top phase, the large bottom phase (including the white interphase) was resuspended in 17.5 ml washing buffer in a 50-ml Falcon tube and repeatedly aspirated with a Pipette Boy. This cell suspension was then centrifuged at 200*g for 10 minutes at 4°C. The pellet was set aside, and to maximize cell yield the supernatant was again repeatedly aspirated and centrifuged. Then, the two pooled cell pellets were resuspended in 1 ml sterile-filtered MACS buffer (5% BSA and 2 mM EDTA in PBS). The cell suspension was incubated with 80 μl myelin removal beads II (Miltenyi Biotec, 130-096-731) for 15 minutes. Meanwhile, a MACS Large Cell Column (Miltenyi Biotec, Bergisch Gladbach, Germany; 130-042-202) was placed in a Magnetic MACS Multistand (Miltenyi Biotec) and activated by allowing 500 μl of MACS buffer to drop through. The cell suspension was passed through the MACS column and the flow-through was collected in a FACS tube pre-coated with 1% sterile-filtered BSA. MACS buffer (500 μl) was then passed through the column twice to increase cell yield. The cell suspension was then incubated with LIVE/DEAD Blue Dead Cell Stain Kit (Thermo Fisher, L23105) for 30 minutes. Immediately afterwards, an a BD Influx FACS machine (86 μm nozzle) was used to sort live tdTomato+ single cells into 96-well skirted PCR plates (Thermo Fisher) containing lysis buffer (containing dNTP [2.5 mM], oligo-dT [2.5 mM], RNAse Inhibitor [Takara Bio, Kusatsu, Japan; 1:40] and ERCC [Ambion, TX, USA; 1:4,000,000]) with the machine set on 'Index Sort' mode to record cell size information and fluorescence parameters used. Plates were immediately frozen on dry ice and stored at -80°C until processing. Genetic recombination (Rbpj deletion in Cx30-CreER+ astrocytes) was induced by intraperitoneal tamoxifen injections for five consecutive days (Sigma-Aldrich, St. Louis, MO, USA; 20 mg/ml in 1:9 ethanol:corn oil; 2 mg/injection). Time points were counted from the last day of injection. Mice with no Rbpj deletion (for isolation of ground state astrocytes) were sacrificed one day after a three-day tamoxifen regimen.  Cells were sorted into lysis buffer (see above) For preparation of single-cell cDNA libraries, the plates were thawed at 72°C for 4 minutes and immediately placed on ice. A reverse transcription of mRNA was performed and the resulting cDNA libraries were immediately subject to amplification for 19 cycles according to the Smart-Seq2 protocol31. The final libraries were subjected to bead purification (AMPure, Beckman Coulter, Brea, CA, USA; 0.6:1 bead-to-sample ratio) to remove excess primers and primer dimers. Library quality was checked using an Agilent Bioanalyzer and DNA High Sensitivity microfluidics chips. After determining that libraries were of sufficiently high quality for downstream sequencing, 0.5-1.5 ng of cDNA from each single-cell library was used for tagmentation reactions with a custom Tn5 enzyme/oligo (comparable to the Nextera XT protocol)32. Nextera XT dual indexes were used (96 index kit) and each library was subjected to an additional 10 cycles of amplification. The final products were again subjected to bead cleaning to remove excess primers and monitored for successful amplification by random sampling of different wells and visualization of library size and quality on the Bioanalyzer. Successful tagmentation reactions were pooled as individual 96-well plates.
attributes.tag: Experimental Factor: genotype
attributes.value: Cx30-CreER; R26-tdTomato; Rbpj(fl/fl)
attributes.tag: Experimental Factor: time
attributes.value: 28
attributes.tag: Experimental Factor: single cell identifier
attributes.value: P3108_1386
attributes.tag: Experimental Factor: organism part
attributes.value: somatosensory cortex