GSM5668575: all_ULA-MK-s009; Mus musculus; RNA-Seq
Proliferating SVZ PLIN2-GFP NSPCs were trypsinized and incubated for 10min at 37°C with the cell viability dye Calcein Red (C34851 ThermoFisher, 1:1000 in 1x PBS). Cells were washed twice with PBS and kept in EDTA-DPBS (E8008, Merck Millipore) at 4°C during the sorting process (Sort-seq). SORT-seq: Single-cell mRNA sequencing was performed at Single Cell Discoveries (single cell sequencing service, the Netherlands) according to an adapted version of the SORT-seq protocol, with previously described primers. Equal numbers of NSPCs with the 25% highest and lowest GFP signal were collected into 384-well plates containing 384 primers and mineral oil (Sigma). After sorting, plates were snap-frozen on dry ice and stored at -80° C. For amplification cells were heat-lysed at 65° C followed by cDNA synthesis using steps based on CEL-Seq2 protocol After second strand cDNA synthesis, barcoded material was pooled into libraries of 384 cells and amplified using IVT. Following amplification, library preparation was processed according to CEL-seq2 protocol where libraries were finally multiplexed using TruSeq small RNA primers (Illumina). Paired-end sequencing was achieved on Illumina Nextseq 500 (Read 1: 26 cycles, index read: 6 cycles, Read 2: 60 cycles).