GSM3272307: MADRK27M012618; Mus musculus; RNA-Seq
Brains were collected in PBS and tumors where microdisected, mechanically dissociated and further enzimatically digested to obtain a single cell suspension. GFP+ tumoral cells were FACS sorted and resuspended in neurobasal media to be processed for 10X single cell RNA-seq Single-cell RNA-seq libraries were prepared per the Single Cell 3′ v2 Reagent Kits User Guide (10x Genomics, Pleasanton, California). Cellular suspensions were loaded on a Chromium Controller instrument (10X Genomics) to generate single-cell Gel Bead-In-EMulsions (GEMs). GEM-RT were performed in a Veriti 96-well thermal cycler (Thermo Fisher Scientific, Waltham, MA). After RT, GEMs were harvested and the cDNAs were amplified, cleaned up with SPRIselect Reagent Kit.  Indexed sequencing libraries were constructed using Chromium Single-Cell 3′ Library Kit for enzymatic fragmentation, end-repair, A-tailing, adapter ligation, ligation cleanup, sample index PCR, and PCR cleanup. The barcoded sequencing libraries were quantified by quantitative PCR using the KAPA Library Quantification Kit (KAPA Biosystems, Wilmington, MA). Sequencing libraries were loaded on a NextSeq500 (Illumina, San Diego, CA) with a custom sequencing setting (26bp for Read 1 and 98bp for Read 2), to obtain a sequencing depth of 100K~200K reads per cell. 10X single cell RNA-seq