GSM6519869: Postnatal Cre+ RNA; Mus musculus; RNA-Seq
To perform single cell Multiome experiment fresh cortical tissue was dissected from 3 different animal for each experimental condition (NESTIN::CRE + and controls) and time point (e14.5 and Postnatal day2). Cortical tissue was then digested using Adult Brain Dissociation kit, mouse and rat (Milteny Biotech, 130-107-677). Tissue was first enzymatically digested with papain and Dnase provived by the kit at 37° for 10', then mechanically dissociated using a p1000 for 10-20 passages, then digested for another 10' and 37°. Afterwards a final mechanical dissociation passage was perform using p200 for 10-20 times. Single cells were then filtered trough a 70nm cell strain to remove debris and centrifuge at 1500 rpm for 5' at room temperature.   After cell counting, cells deriving from the 3 different animals for each experimental condition were pulled together and nuclei were extracted following 10x Genomics protocol for Nuclei Isolation from Embryonic Mouse Brain Single Cell Multiome. A total of 1 million cells for each experimental condition were used as a starting point for nuclei extraction. After nuclei counting single cell libraries were prepared using Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle (10x Genomics, cod. FC51000285). For embryonic samples 9000 cells and 14000 cells for postnatal samples, were loaded on Chromium controller after Tn5 trasposition for single cell library preparation.   For each experimental timepoint a unique RNA-seq and ATAC-seq library was obtained and sequenced on a Nova-seq6000 platform at San Raffaele hospital omics facility transcriptomic single cell 10x multiome