title: GSM6886288: Glast, replicate 1, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Primary ECs and NSPCs from mouse brain cortex and SVZ were FACS-isolated.Briefly, brain cortex and SVZ were harvested , minced into small pieces . Minced pieces were then subjected to enzymatic dissociation in collagenase/dispase  solution for 30 min at 37C . The digested tissues were triturated in 2% FBS in PBS supplemented with DNAse  with a P1000 pipette. Antibodies  were added to the resuspended cells  and incubated for 20 mins on ice protected from light. Post-immunostaining cells were washed with HBSS/BSA/glucose buffer. From SVZ CD45- cells, ECs (CD31+Glast- population) and NSPCs (Glast+CD31- population) were collected using a BD FACS Melody cell sorter in pre-filled FACS tubes with EC and NSC media respectively to cushion the cells. Post-FACS processing, cells were pelleted, snap frozen in liquid nitrogen and stored at -80 C for RNA isolation. FACS-purified live single cell suspensions were used for single cell RNA library preparation and next-generation sequencing. Single cell RNA libraries were prepared using the 10X Genomics Chromium Next GEM Single Cell 3' Reagent Kit v3.1 (PN-1000121) and Chromium Controller. Next-generation sequencing was performed using an Illumina NextSeq 2000 Sequencing System on the P3 flow cell with a 100 bp paired-end sequencing reagent kit. Two biological replicate samples for each condition were prepared, sequenced, and aligned using CellRanger v5.0.0 to the mm10 genome. Estimated cell number outputs for each sample were: Cx43fl/fl (1) = 1083; Cx43fl/fl (2) = 1076; Cx43GlastiKO (1) = 710; Cx43GlastiKO (2) = 1319; Cx43ECiKO (1) = 571; Cx43ECiKO (2) = 2674. Sequencing yielded a mean of ~70,000,000 reads/sample, ~78,000 reads/cell, and ~1,500 genes/cell.