GSM8768601: D8_WT63; Mus musculus; RNA-Seq
Total RNA extraction from BMDCs generated as described above was performed using the RNeasy Mini RNA kit (Qiagen) including on-column DNase digestion according to the manufacturer's protocol. The integrity of the RNA was confirmed in BioAnalyzer using RNA 6000 Pico kit (Agilent Technologies) (8.8 < RNA integrity no. (RIN) < 10). RNA-seq libraries were prepared from 300 ng to 1 µg of total RNA using an Illumina TruSeq Stranded mRNA Library Preparation kit and an Illumina Stranded mRNA Prep Ligation kit (Illumina, cat# 20040532), which allowed strand-specific RNA-seq. First, poly(A) selection using magnetic beads was performed to focus sequencing on polyadenylated transcripts. After fragmentation, cDNA synthesis was performed, and the resulting fragments were used for dA-tailing and were ligated to the TruSeq indexed adapters (for the TruSeq kit) or RNA Index Anchors (for the mRNA Ligation kit). PCR amplification was performed to generate the final indexed cDNA libraries (with 13 cycles). Individual library quantification and quality assessment was performed using a Qubit fluorometric assay (Invitrogen) with a dsDNA High-Sensitivity Assay kit and LabChip GX Touch using a High-Sensitivity DNA chip (PerkinElmer). Libraries were then pooled at equimolar concentrations and quantified by qPCR using a KAPA library quantification kit (Roche). Sequencing was performed on a NovaSeq 6000 instrument (Illumina) using paired-end 2 × 100 bp sequencing with an average depth of 30 × 106 clusters per sample.