Zymo quick rna microprep kit

hiPSC-derived astrocytes (iAstrocytes, TCW et al., Li et al., and Krencik et al. astrocytes), NPCs, and iNeurons were cultured in their respective media and treated with vehicle control or IL-1α+TNF+C1q for 24 hours. RNA extraction was then performed with Zymo Quick-RNA Microprep kit (Zymo Research cat. no. R1051). 50–100 ng of RNA was then used to construct bulk RNA-seq libraries using the QuantSeq 3’ mRNA-Seq Library Prep Kit FWD for Illumina (Lexogen cat. no. 015.96) following the manufacturer’s instructions. The concentration of QuantSeq libraries mRNA-seq library was quantified using the Qubit dsDNA HS Assay Kit (ThermoFisher Scientific cat. no. Q32851) on a Qubit 2.0 Fluorometer. Library fragment-length distributions were quantified with High Sensitivity D5000 Reagents (Agilent Technologies cat. no. 5067–5593) on the 4200 TapeStation System. The libraries were sequenced on an Illumina NextSeq 2000 instrument with single-end reads.

One thousand cells were sorted directly into RLT buffer (79216; Qiagen) containing 1% 2-Mercaptoethanol. RNA was isolated using Zymo Quick-RNA MicroPrep Kit (11–328M; Zymo Research). Synthesis of cDNA was performed using SMART-Seq v4 Ultra Low Input RNA Kit (634894; Takara Bio) kit. Final libraries were generated using 200 pg of cDNA as input for the NexteraXT kit (Illumina, FC-131–1024). RNA-seq libraries for naïve B cells and ex vivo generated PC were sequenced at University of Alabama Birmingham Helfin Center for Genomic Sciences on a NextSeq500/550 using 75 bp paired-end chemistry. RNA-seq libraries for PC generated in vivo were sequenced on NovaSeq6000 at Novogene. Raw sequencing reads were mapped to the mm10 genome using STAR v2.5.3a (62 (link)). Raw and reads per kilobase per million (Rpkm) normalized gene expression counts were determined by annotating the coverage across all exons for all unique ENTREZ genes using the GenomicAlignments (63 (link)) package. Differentially expressed genes are listed in Supplemental Table I. analysis was performed using edgeR (64 (link)) with FDR ≤ 0.05 being considered significant. Differentially expressed genes are Gene Set Enrichment Analysis (GSEA) (65 (link)) was performed using a pre-ranked gene list generated by multiplying the sign of the fold change (negative or positive) by the −log₁₀ of the edgeR derived p value.