Illumina HiSeq 1500 sequencing; PPARD in MDM - chipseq
PPARD in MDM - chipseq
Cells were isolated on October 14th 2014 from two samples of healthy human donor blood of unknown gender via LSM1077 gradient centrifugation. Adherent cells were positivly selected on primaria 6 well dishes (3x10^6 monocytes per well in 2 ml PBS (PAA)) followed by 6 washing steps with PBS. Finally the monocytes were cultured for 7d in humidified incubator at 37 °C, 5 % CO2 in 2ml RPMI medium supplemented with 10 % FCS (both PAA). Lysis in hypotonic lysis buffer Nuclei were resuspended in ChIP RIPA and sheared with a Branson S250D using a microtip (60 pulses of 1 s, 2 s pause, 20% amplitude) with coolling (ice-ethanol). A 15 min 20,000xg supernatant was used for IP (after preclearing). The Diagenode Microplex Library Preparation kit (Diagenode, C05010011) was used according to the manufacturer's instructions with 1-2 ng of input DNA. DNA concentrations were measured on a Nanodrop 3300 using the Quant-It dsDNA High-Sensitivity Assay Kit (Life Technologies).