GSM2474649: G05_T03_D; Homo sapiens; RNA-Seq
At the indicated time points, cells were collected by centrifugation, washed twice with PBS and lysed in RLT buffer supplemented with beta-mercaptoethanol. Lysates were homogenized using QiaShredder columns and stored at -80°C until processing. RNA and proteins were extracted with the AllPrep DNA/RNA/Protein Mini Kit, with the following modifications. Centrifugations were carried out at 9600 x g except protein precipitation at 20800 x g. RNA was eluted with 40 µl RNase-free water and elution was repeated with the first eluate. An aliquot of RNA was taken for nanodrop, bioanalyzer and qRT-PCR, RNA was frozen on dry ice and stored at -80°C. One microgram of RNA per sample (except for the 2h time point samples of Donor 2: 500 to 900 ng) was used for library preparation with the TruSeq Stranded mRNA HT kit (Illumina), with Dual Indexing adapters (8-plex). As control, an ERCC Spike-In Control Mix was used. Libraries were purified from excess sequencing adapters with Agencourt AMPure XP beads. Library concentration was measured on a Qubit 2.0 Fluorometer and library quality and size were determined by using an Agilent High Sensitivity DNA Kit on a 2100 Bioanalyzer. Libraries were quantified with the KAPA Library Quantification Kit.
GEO Accession
GSM2474649