title: GSM6640461: IFNg-treated batch 3; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was isolated from macrophages exposed to stimulus for 24h (n=4-6 wells per condition) using the Micro RNeasy kit (Qiagen) according to the <100,000 cells protocol. RNA quality and integrity was assessed on a 2100 BioAnalyser Picochip (Agilent Technologies). mRNA was converted into libraries of double stranded cDNA molecules as a template for high throughput sequencing following the SMART-Seq2 protocol as described by Picelli et al. Shortly, mRNA was primed for SMART reverse transcription from 5 ng of total RNA using poly-T oligos. Resulting cDNA was pre-amplified by SMART ISPCR. Fragmentation was carried out using the Illumina Nextera XT kit, followed by PCR amplification and indexing. Size selection and purification of library fragments with preferentially 300-400 bp in length was performed using SPRIBeads (Beckman-Coulter). The size-distribution of cDNA libraries was measured using the Agilent high sensitivity D5000 assay on a Tapestation 4200 system (Agilent). cDNA libraries were quantified using a Qubit high sensitivity dsDNA assay. 75 bp single-end sequencing was performed on a NextSeq500 system using High Output v2.5 chemistry.