Total RNA from iPSC and iPSC-derived microglia was extracted using RNeasy Plus kit (QIAGEN, catalog #74136). Library construction and sequencing were performed by Novogene. The mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either deoxyuridine triphosphate nick end labeling for a directional library or 3′-deoxythymidine 5′-triphosphate for the nondirectional library. The nondirectional library was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was prepared through end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification steps. The library was checked with Qubit and real-time polymerase chain reaction for quantification and bioanalyzer for size distribution detection. Quantified libraries were pooled and sequenced on Illumina NextSeq500 platforms by Novogene. Approximately 40 to 60 million paired reads were generated for each sample (table S3). Reads were aligned to the hg38 human reference genome by HISAT2 (v.2.1.0) (96). Transcript counts were extracted using the featureCounts function of the Rsubread package. Details of the bioinformatics analysis methods for RNA-seq data are provided in the Supplementary Materials.
