GSM5124122: Sample_SS4_old_69; Mus musculus; RNA-Seq
Homozygous Nestin:CFPnuc mice (Encinas et al., 2006) (Strain: B6.Cg-Tg(Nes-CFP)1Enik/J) were used for all single-cell RNA-seq experiments (Shin et al., 2015). Mice were euthanized by cervical dislocation, and brains were immediately immersed into cold Hibernate A solution (BrainBits). Dentate gyri were dissected under a stereomicroscope as previously described (Hagihara et al., 2009). All procedures were performed with approved protocols in accordance with institutional animal guidelines. The single cell suspension was prepared by using Neural Tissue Dissociation Kit (P), with the addition of one cleaning step with Percoll (1:10 dilution) to remove myelin layer and cellular debris. Propidium Iodide was added to determine cell viability and cells were sorted on a BD FACs Aria II with a 70μm nozzle at 13 psi (Figure S3). Single cells were collected onto the ice-cold 8-sample parafilm-covered glass slides with 1.25uL of 1X lysis mixture (Clontech Laboratories SMART-Seq v4 Ultra Low Input RNA Kit for sequencing) and immediately transferred in individual 0.2-ml RNase-free 8-well strip (Figure S3).  To avoid any effects due to the FACs sorting protocol, cells were kept on ice the entire procedure, and the actual collection was performed at 4°C. cDNA was generated using the SMART-Seq v4 Ultra Low Input RNA Kit (Clontech Laboratories) for sequencing with modifications to bring down the cost. Briefly, all reagents from the kit (for Reverse transcription and cDNA amplification) were miniaturized 10-fold by utilizing FACs collection in a very low volume of the lysis buffer. The same quality of cDNA was achieved as for the original kit. The amplification product was purified using Ampure XP beads according to the manufacturer's protocol. Quality control and validation were performed by using qPCR for Actin expression and Agilent TapeStation 4200 (Agilent Technologies). The amplification product was sheared using library preparation Nextera XT Kit (Illumina Inc.) following the manufacturer's protocol. In brief, the purified cDNA was tagmented and fragmented using a miniaturized protocol. cDNA fragments were barcoded using i5 and i7 indices (Illumina Inc.) and the product was purified using Ampure RNA clean beads. The purified ligation product underwent 13 cycles of PCR. A Mantis Liquid Handler (Formulatrix, Inc.) was used to handle low volume pipetting. The libraries were multiplexed and sequenced using Illumina NextSeq 550 (Illumina Inc.) 75 paired end run. To minimize the batch effect on the sequencing all cells were loaded into one chip.