GSM9207335: P12, Mouse Brain, Rostral migratory stream; Mus musculus; RNA-Seq
For the P6 and P12 ages, mice (2 males and 2 females for each timepoint) were anesthetized with isoflurane and then perfused intracardially with 0.9% saline. Brains were removed and the RMS was carefully micro-dissected from the rest of the brain. The RMS is a relatively narrow pathway; therefore, our data likely includes some cell types from regions immediately adjacent to the RMS. RMS tissue was dissociated according to the 10X Genomics Chromium sample preparation protocol. Tissue was cut into smaller pieces and placed into 2mL papain solution (BrainBits PAPHE, NC0435282). Tissue was incubated at 37oC for 20 minutes, the papain solution was removed and 2mL of Hibernate E medium (BrainBits HE500, NC9063748) was added to the samples and the sample was triturated with a fire-polished 9-inch Pasteur pipette. The sample was then set aside for 1 minute to allow for the tissue debris to settle. Cells in the supernatant were removed and passed through a 70µm mesh filter (Electron Microscopy Sciences, 6475200) two times. Cells were centrifuged for 2 minutes at 200rcf., the supernatant discarded, the cells resuspended in 1mL of NbActiv1 medium (BrainBits NB1100, NC1482275) and passed through a 70µm mesh filter one final time before being placed on ice. Cells were stained with DAPI (1µg/mL) for 5 minutes and live (DAPI-negative) singlets were sorted using Aurora Cell Sorter (Cytek) with a 100µm nozzle at low pressure. Sorted live cells were collected into NbActiv1 medium (BrainBits NB1100, NC1482275). For our P60 sample, the same harvesting and microdissection procedures were followed. However, tissue dissociation was performed using the Singulator 200 (S2 Genomics) following the manufacturer's protocol (Cell Isolation from Fresh Tissue for Single Cell Sequencing Applications, S2 Genomics). Cell viability and concentration were assessed using a Bio-Rad TC20 Automated Cell Counter and loaded onto 10X Genomics Chromium System. Libraries were generated with the 10X Chromium Single Cell 3′ v2 reagent kit according to the manufacturer's instructions and sequenced on an Illumina NextSeq 550. Prepared cells were processed for single cell transcriptome analysis using the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1 (10x Genomics, Pleasanton, CA, USA).  Cells were diluted to the appropriate concentration per manufacturer recommendations prior to ensure proper loading.  GEM reverse transcription, cDNA amplification and library generation followed manufacturer's protocol (10x Genomics, Pleasanton, CA, USA).  Briefly, cells are captured in emulsion and reverse transcription is performed while simultaneously barcoding individual cells.  After RT, cDNA is released from the GEMs and amplified.  cDNA is used as an input for library preparation where its first fragmented, size selected and then ligated with Illumina P5 and P7 adapter ends, using unique dual index barcodes for each sample.