Sample_CMML1900-Input
Protocols: Peripheral blood mononucleated cells (PBMC) were obtained from healthy donor buffy coats (Etablissement Français du sang, Rungis, France) or CMML whole blood, separated on Pancoll (Pan-Biotech, Dutscher, Brumath, France). Peripheral blood CD14+ monocytes were sorted with magnetic beads and the AutoMacs system (Miltenyi Biotech, Paris, France). Cells were cross-linked with addition of 1% formaldehyde directly to the culture medium for 10 min at RT with agitation. Fixation was stopped by addition of 125mM glycin during 5 min at RT with agitation and samples were then washed 2 times in ice-cold PBS before addition of SDS lysis buffer (Millipore, 10uL per 1.106 cells) supplemented with 1% protease inhibitor cocktail (Active Motif). Samples were vortexed and incubated 15 min on ice before 10 min sonication at 40W (Covaris S220, Woodingdean, UK). The chromatin immunoprecipitation was carried out using ChIP-it express kit according to manufacturer's instruction (Active Motif) with a polyclonal anti-EGR1 antibody (sc-110, santa cruz bioechnology). Enriched DNA from ChIP and Input DNA fragments were end-repaired, extended with an 'A' base on the 3′end, ligated with indexed paired-end adaptors (NEXTflex, Bioo Scientific, Proteigene, Saint Marcel, France) using the Bravo Platform (Agilent, Les Ulis, France), size-selected after 4 cycles of PCR with AMPure XP beads (Beckman Coulter, Villepinte, France) and amplified by PCR for 10 cycles more.
ENA first public
2021-11-15
ENA last update
2021-11-15
External Id
SAMEA10792501
INSDC center alias
INSERM U1287
INSDC center name
INSERM U1287
INSDC first public
2021-11-15T00:13:41Z
INSDC last update
2021-11-15T00:13:41Z
INSDC status
public
Submitter Id
E-MTAB-11132:Sample_CMML1900-Input
age
73
broker name
ArrayExpress
cell type
monocyte
common name
human
developmental stage
adult
disease
chronic myelomonocytic leukemia
individual
CMML1900
organism part
blood
sample name
E-MTAB-11132:Sample_CMML1900-Input
sex
male