GSM9683944: 1-spr-exphx-OPNp; Mus musculus; RNA-Seq
After 48h in co-culture, 1x Brefeldin A (BioLegend, #420601) was added. Splenocytes were collected for FACS analysis and RNAseq analysis. RNA was extracted using RNAeasy Mini Plus Kit (Qiagen, #74134) and quantified in LabChip GX Touch Nucleic Acid Analyzer (PerkinElmer). RNA quality was determined by Agilent's 2100 Bioanalyzer Lab-Chip technology. Only samples with RNA Integrity Number (RIN)>7 was used for sequencing. Total RNA samples (300ng; with average RIN/RQS 7.5, range 2.5-9.5) were converted into sequencing libraries using the "QuantSeq 3' mRNA-Seq V2 Library Prep Kit (FWD) for Illumina" (Lexogen, Cat.No. 191). Briefly, library generation is initiated by reverse transcription with oligodT priming, followed by a random-primed second strand synthesis. A UMI Second Strand Synthesis module was used, in which random primers featuring a 6 nt long Unique Molecular Identifier tag are used {to be addressed for bias correction at data analysis}. Directional cDNA libraries, stranded in the sense orientation {--library-type fr-secondstrand in TopHat, --stranded=yes in HTSeq, --rna-strandness F in HISAT}, were completed by PCR and sequenced in a single read format on an NovaSeq X Illumina instrument.