GSM7082374: iMG, rep2; Homo sapiens; RNA-Seq
For iMGs, cells were carefully manually scraped from the Matrigel coated plate and were concentrated by a 300 rcf x 5 minutes. For oMGs, organoids were carefully collected, allowed to settle by gravity, and then dissociated mechanically in staining buffer (HBSS 1x with 1mM EDTA and 1% BSA) using a 2 ml polytetrafluoroethylene pestle. iMGs and oMGs were stained with the following Biolegend antibodies/dyes: CD64-APC (305014), CX3CR1 PCP-Cy5.5 (341614), CD14-488 (325610), CD11b-PE (301309), HLADR PE-Cy7 (307616), CD45-APC-CY7 (368516), and Zombie Violet (1:1000, Biolegend) for live/dead discrimination. xMGs were isolated and sorted at indicated time points following xenotransplantation. Briefly, mouse brains were homogenized as previously described by gentle mechanical dissociation (Gosselin et al., 2014) and stained with DAPI, CX3CR1-APC (Biolegend 149008), anti-human CD11b-PE (Biolegend 301306), anti-human CD45-FITC (Biolegend 368508). Microglia were purified with either a BD Influx  or FACS  AriaFusion.TA RNA-seq libraries were prepared as previously described (Gosselin et al., 2017). Briefly, mRNAs were incubated with Oligo d(T) Magnetic Beads (New England BioLabs S1419), then fragmented in 2x Superscript III first-strand buffer (Thermo Fisher Scientific with 10mM DTT (Thermo Fisher Scientific 18080044) at 94oC for 9 minutes. Fragment mRNA was then incubated with 0.5 μl of Random primers (3 mg/mL) (Thermo Fisher Scientific 48190011), 0.5 μl of 50mM Oligo dT primer, (Thermo Fisher Scientific, 18418020), 0.5 μl of SUPERase-In (Thermo Fisher Scientific AM2694), 1 μl of dNTPs (10 mM) at 50°C for one minute. Then, 1 μl of 10mM DTT, 6 μl of H2O+0.02%Tween-20 (Sigma), 0.1 μl Actinomycin D (2 mg/mL), and 0.5 μl of Superscript III (Thermo Fisher Scientific) were added to the mixture. cDNA was then generated by incubating mixture in a PCR machine at the following conditions: 25oC for 10 minutes, 50°C for 50 minutes, and a 4°C hold. Product was purified using RNAClean XP beads (Beckman Coulter A63987) according to manufacturer's instructions and eluted with 10 μl of nuclease-free H2O. Eluate was then incubated with 1.5 μl of Blue Buffer (Enzymatics P7050L), 1.1 μl of dUTP mix (10 mM dATP, dCTP, dGTP and 20 mM dUTP), 0.2 mL of RNase H (5 U/mL Y9220L), 1.2 μl of H2O+0.02%Tween-20, and 1 μl of DNA polymerase I (Enzymatics P7050L) at 16°C overnight. DNA was then purified using 3 μl of SpeedBeads (Thermo Scientific Fisher 651520505025) resuspended in 28 μl of 20% PEG8000/2.5M NaCl to final of 13% PEG. DNA eluted with 40 mL nuclease free H2O+0.02%Tween-20 and underwent end repair by blunting, A-tailing and adaptor ligation (Heinz et al., 2010a) as previously described using barcoded adapters. Libraries were PCR-amplified for 12-15 cycles, size selected by gel extraction for 200-500 bp, and sequenced on a HiSeq 4000 (Illumina) or a NextSeq 500 (Illumina) for 51 cycles.