GSM6939555: SVZ_3; Mus musculus; RNA-Seq
8 male Envigo C57BL/6J  mice per experiment were used to sort cells from the SVZ. Using a modified previously published protocol for dissection, SVZ tissue was dissected into cold HBSS without Ca2+ and Mg2+, followed by mincing of the tissue with a razor blade. Tissue was resuspended in 0.05% Trypsin-EDTA, and put in a 37 degrees C water bath for 6 minutes. Trypsin was quenched using a double volume of pre-warmed trypsin inhibitor including DNAse, and centrifuged for 300xg for 5 minutes. The pellet was resuspended in a cold, sterile-filtered, 0.5% BSA/PBS solution (sort buffer) and dissociated by pipetting 20 times up/down. A 45µM mesh filter was pre-wet with the same solution, and the run-through discarded. The brain sample was run through the filter, followed by a 1ml rinse. The cell solution was spun for 300xg for 5 minutes for 4 degrees C. All spins and treatments from this point forward were performed at 4 degrees C.  For 2 of the 3 experiments, myelin was removed using a Miltenyi Magnetic sorting protocol. In short, the appropriate volume of cold sort buffer was added based on weight, as well as myelin removal beads, followed by incubation for 15 minutes in the refrigerator. After washing, lysate was centrifuged at 300xg for 10 minutes. The pellet was resuspended in sort buffer, and then was filtered through the magnetic column. The effluent was spun down, and then resuspended in sort buffer and an anti-GLAST-Biotin antibody (Miltenyi), incubating for 10 minutes in the refrigerator. Following wash and centrifugation, cells were further labeled with anti-biotin microbeads (Miltenyi) for 15 minutes in the refrigerator. To prepare cells for loading onto the magnetic column, they were washed, centrifuged, and resuspended in sort buffer. Cells that were GLAST+ were on beads that remained in the column on the magnetic plate. The column was washed, and beads and cells eluted into a collection tube using a plunger once removed from the magnetic plate, centrifuged, and resuspended in an ice-cold staining buffer containing L-15, 1M HEPES 7.4 pH, 100x Pen-strep, and 100mM EDTA. CD24-APC-eFluor780 (M16947-0242-82; 1:200) and O4-Cy7 (Miltenyi – 130-115-810; 1:200) were added for a total of 45 minutes, with CD133-APC (Clone 13A6; 1:50) added in the final 25 minutes of incubation on ice. Cells were then washed and centrifuged, and resuspended in FACS buffer with 4µg/ml DNAse, and transported to the FACS machine on ice. There, using previously established compensated settings, GLAST+ (per bead sorting) CD133+ (low to middle intensity) CD24-, O4- (together labeled in the same “dump” channel) were sorted into a 384 well plate, with autofluorescence intensity measured for each cell. To establish the compensated settings, cells collected from the brain were singly labeled for each antibody, and then as a group. Viable single cells were FACS sorted into 384-well plates, snap frozen, and sent to Single Cell Discoveries for processing and analysis.  This experiment was performed 3 separate times, collecting cells from each experiment to be analyzed together. Libraries for each plate were prepared according to an adapted version of the SORT-seq protocol (Muraro et al. 2016 with primers described in van den Brink et al. 2017). Briefly, after cell lysis, second-strand cDNA synthesis was done, and all the barcoded material from one plate was pooled into one library and amplified using in vitro transcription (IVT). Following amplification, library preparation was done following the CEL-Seq2 protocol (Hashimshony et al. 2016)  to prepare a cDNA library for sequencing using TruSeq small RNA primers (Illumina). The DNA library was paired-end sequenced on an Illumina Nextseq™ 500, high output, with a 1×75 bp Illumina kit (read 1: 26 cycles, index read: 6 cycles, read 2: 60 cycles)