GSM9251039: monoculture astrocyte A; Homo sapiens; RNA-Seq
To dissociate miBrains, samples were washed with PBS, then were incubated for 5-10 minutes in a mixture of accutase, collagenase IV (1:20), and dextranase (1:20). These were triturated 15-20 times with a P1000 pipette to form a homogeneous suspension, then pelleted and washed with DPBS twice. Astrocytes, iMG, and neuron monocultures grown in parallel were dissociated using accutase, then washed with DPBS prior to proceeding with antibody labeling and sorting. Oligodendroglia were fixed with 4% PFA on ice for 20 minutes prior to dissociation to ensure stability. iMG were incubated with CD45-Cy7 alongside isotype controls at 1:200 on ice for 25 minutes before proceeding with sorting. Dissociated miBrains and monocultures were resuspended in FACS buffer (DPBS, BSA, EDTA) with DAPI and filtered through a 40 μm strainer. Samples were sorted on a BD FACSAria equipped with a 70 μm nozzle. Neurons were FACS-isolated based on tdTomato expression, astrocytes and oligodendroglia based on mCherry, and iMG based on CD45 expression. RNA was harvested in triplicate and in parallel from 20K neurons, 50K astrocytes, 50K oligodendroglia, or 2K CD45-positive iMG per replicate. Cells were gated on FSC-A vs SSC-A to separate intact cells from debris, then FSC-H vs FSC-W and SSC-H vs SSC-W to separate single cells from doublets, then DAPI to exclude dead cells, and then the channel corresponding to the antibody used. Cells were sorted into 96-well plates containing 50 μl RNAprotect per well. For live samples, RNA was isolated from sorted cells using the Qiagen RNeasy isolation kit according to the manufacturer's protocol. For fixed samples, sorted cells were spun down and resuspended in 200 μL digestion buffer (RecoverAll kit) supplemented with 1:50 Proteinase K, incubated at 50°C for 15 minutes, and then at 80°C for 15 minutes with no agitation to de-crosslink RNA from fixed tissue. 800 μL Trizol was added to the sample and incubated for 5 minutes, then 215 μl chloroform was added and the sample was vortexed vigorously for 30 seconds. Sample was transferred to a 5-prime phase lock heavy gel tube and centrifuged for 15 minutes at 12,000 g. The upper layer containing RNA was transferred to a fresh 1.5 ml microcentrifuge tube and an equal volume of 100% ethanol before proceeding with RNA isolation using Zymo Direct-zol RNA microprep kit according to the manufacturer's protocols. Standard RNA quality control was performed using AATI fragment analyzer. RNA sequencing libraries were prepared using NEB Ultra II PolyA stranded library and sequenced using Illumina NextSeq500.