Bulk RNA-Seq of HMC3 microglia treated with drugs driving the Disease-associated microglial (DAM-) signature

In brief, 0.75–1×10⁶ HMC3 microglial cells were seeded into a 6.5cm or 10cm dish and incubated o.n. The next day, microglia were treated with the respective concentrations of Vorinostat (1μM; Ambeed; Cat #: A234507), Entinostat (10μM; Ambeed; Cat #: A122285) or DMSO (Sigma-Aldrich, Cat #:472301) as control and incubated for 24 hrs before harvest. Cells were trypsinized (Gen Clone; Cat #:25–510F), counted, the cell viability was assessed. Cells were then washed with ice-cold PBS (Corning, Cat #:21–040–CV) and resuspended in 350μl RLT Lysis buffer (Qiagen; Cat #: 74136) containing 2-Mercaptoethanol (Thermo Fisher Scientific, Cat #:63689), and isolated using Qiagen Plus Mini Kit (Qiagen; Cat #: 74136). RNA quality was assessed using the TapeStation (Agilent) prior to further processing for RNA sequencing. mRNA libraries were prepped using poly-A pull-down to enrich mRNAs from total RNA samples followed by Illumina TruSeq Stranded mRNA Library prep (Illumina, Cat#: 20020595), in accordance with manufacturer recommendations, and using IDT for Illumina TruSeq DNA UD Indices (Illumina, Cat#: 20022370) for adapters. Briefly, 500ng of total RNA was used for purification and fragmentation of mRNA. Purified mRNA underwent first and second strand cDNA synthesis. cDNA was then adenylated, ligated to Illumina sequencing adapters, and amplified by PCR (using 10 cycles). The cDNA libraries were quantified using Fragment Analyzer 5300 (Advanced Analytical) kit FA-NGS-HS (Agilent, Cat#: DNF-474–1000) and Spectramax M2 (Molecular Devices) kit Picogreen (Life Technologies, Cat#: P7589). Libraries were sequenced on an Illumina NovaSeq6000 sequencer at Columbia Genome Center. We multiplex samples in each lane, which yields targeted number of paired-end 100bp reads for each sample. We used RTA (Illumina) for base calling and bcl2fastq2 (version 2.20) for converting BCL to fastq format, coupled with adaptor trimming. We performed a pseudoalignment to a kallisto index created from transcriptomes (Ensembl v111, Human:GRCh38.p14; Mouse:GRCm39; mRatBN7.2) using kallisto (0.50.1). The references and kallisto version were updated on April 29, 2024 to ensembl v111 and kallisto 0.50.1.