GSM2653913: KO monocytes 2; Mus musculus; RNA-Seq
Total RNA from sorted monocytes and from monocytes cultured with GM-CSF and IL-4 for 1 and 2 days (2 samples per group from 2 independent experiments) was isolated using Trizol as recommended by the manufacturer (Invitrogen), and then further purified using RNeasy columns (Qiagen). Presence of contamination with chemicals was assessed by Nanodrop spectrophotomer using 260/280 and 260/230 ratios. Quantification of total RNA was made by QuBIT (Life Technologies). Library preparation was done with the KAPA mRNAseq stranded library preparation kit (Roche, after mRNA capture with Dynabeads® mRNA DIRECT™ Purification Kit). 20 ng of total RNA were used as input for library preparation. Ligation was made with 0.5µl (final concentration 1.8 nM) of NEBNext Index primer for Illumina and 16 PCR cycles were required to amplify cDNA libraries. Libraries were then quantified by QuBit and the quality assessed with BioAnalyzer DNA 1000 and/or DNa High sensitivity chip. All librairies were diluted to 10nM and normalized by qPCR using the KAPA library quantification kit (KAPA). Libraries were pooled to equimolar concentration.
GEO Accession
GSM2653913