title: GSM3457270: P28 WT Astrocyte rep 2; Mus musculus; RNA-Seq
library_construction_protocol: Astrocytes were acutely isolated as previously described (Holt and Olsen, 2016; Kahanovitch et al., 2018; Stoica et al., 2017). Following dissociation, microglia and myelin were first removed from the cell suspension. Cells were incubated for 15 minutes at 4°C with 15µL of Miltenyi Biotec's Myelin Removal Kit and Cd11b+ MicroBeads. The suspension was then applied to a prepped LS column, washed three times, and the flow-through collected. This flow through was subsequently used to isolate astrocytes utilizing Miltenyi Biotec's ACSA-2+ MicroBead kit. The cell suspension (in 150uL 0.5% fatty-acid free BSA in PBS) was incubated at 4°C for 15 minutes with 15-20µL FcR blocker, followed by a 15 minute incubation with 15-20µL ACSA-2 microbeads.  Cells were applied to a prepped LS column. Astrocytes were eluted from the LS column after three washes, with 5mL buffer and the supplied plunger. Total RNA was isolated using Ambion's PureLink RNA Mini Isolation kit according to the manufacturer's instructions. RNA samples designated for RNA Sequencing were eluted in 30 µL filtered, autoclaved Mill-Q water. RNA samples were tested for quality on the Agilent Tapestation 2200 (Agilent Technologies, Santa Clara, CA). The NEB Next rRNA Depletion Kit (NEB #E6310X) was used to process 250 ng of total RNA. RNA-Seq libraries (400 bp) were created using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (NEB #E7760L). Samples were individually indexed using the NEBNext Multiplex Oligos for Illumina (NEB #E6609S). Adapter ligated DNA was amplified in 13 cycles of PCR enrichment. Libraries were quantified with the Quanti-iT dsDNA HS Kit (Invitrogen) and qPCR. Library validation was performed on the Agilent 2200 Tapestation. Independently indexed stranded cDNA libraries were pooled and sequenced for 150 cycles with the Illumina NovaSeq 6000 S2 Kit. All samples were sequenced at 85-90 million read depth, paired-end 2 x 150 bp, and in reverse-stranded orientation.