title: GSM8335712: cortex 3-weeks replicate 3 (028); Mus musculus; RNA-Seq
library_construction_protocol: Freshly collected brain cortical and hippocampal tissues from female WT C57Bl/6J were washed with 1ml of 1× Hanks's balanced salt solution without phenol red, Ca2+ and Mg2+ (HBSS). White matter was removed to the possible maximum, before the tissue was chopped into small pieces using scalpel and transferred into conical tubes containing 1 ml of an enzymatic mixture of dispase II (0.1%), papain (0.01%) and DNase I (0.05%) made in 1× HBSS containing 12.4 mM magnesium sulfate (Si6). Tubes were incubated at 37ºC for 10 min without agitation. The enzymic activity was stopped by adding 4 ml of 20% ice-cold FBS solution made in 1× HBSS. The cell suspension was centrifuged at 500× g at 4 ºC for 5 min. For myelin removal, the cell pellet was resuspended in 1 ml of 20% Percoll® solution made using 10× phenol red HBSS and 1× HBSS. After complete resuspension of the cell pellet, an additional 4 ml of the 20% Percoll® solution was added, bringing the volume up to 5 ml. The suspension was then gently overlaid with 5 ml of 1× HBSS. Tubes were spun at 500× g at 4ºC for 10 min with no break. The supernatant containing the myelin debris was carefully discarded, and the cell pellet was resuspended in 1 ml flow cytometry buffer. At this step, samples from the three animals were pooled into one 15 ml conical tube and centrifuged at 500× g at 4ºC for 5 min.  The supernatant was carefully discarded, and the cell pellet was resuspended in 500 μl flow cytometry buffer. The cell suspension was transferred into a 1.5 ml microtube. Cell viability was accessed using the trypan blue exclusion method and processed for single-cell RNA sequencing. Isolated cells were processed for single-cell RNA sequencing at the eukaryotic single-cell genomics facility (ESCG) at Karolinska Institutet, Sweden using the 10x Genomics Chromium method (version 3.0.1) and sequenced using Illumina NovaSeq 600 with an S2 flow cell.