Illumina NovaSeq 6000 paired end sequencing; ChIP-seq of CSF-1R in THP-1 cell lines deleted for EGR1 gene by CRISPR-Cas9 and ChIP-seq of CSF-1R in 2 CMML patients
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
No description
Antigen
NA
Cell type
Cell type Class
Blood
Cell type
THP-1
Primary Tissue
Blood
Tissue Diagnosis
Leukemia Acute Myelogenous
Attributes by original data submitter
Sample
ENA first public
2019-03-28
ENA last update
2019-03-12
ENA-CHECKLIST
ERC000011
External Id
SAMEA5417267
INSDC center alias
Inserm U1170
INSDC center name
Inserm U1170
INSDC first public
2019-03-28T17:02:37Z
INSDC last update
2019-03-12T09:39:31Z
INSDC status
public
Submitter Id
E-MTAB-7756:THP-1 WT#2/clone 31 CFMS
broker name
ArrayExpress
cell line
THP-1
cell type
monocyte
common name
human
disease
acute monocytic leukemia
genotype
wild type genotype
organism part
blood
sample name
E-MTAB-7756:THP-1 WT#2/clone 31 CFMS
Sequenced DNA Library
library_name
THP-1 WT#2/clone 31 CFMS_p
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
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). THP-1 cells were cultured in RPMI 1640 Glutamax medium (ThermoFisher Scientific) supplemented with 10% heat inactivated fetal bovine serum (FBS, Lonza, Amboise, France), 1% penicillin/streptomycin and 2mM L-Glutamine (ThermoFisher Scientific) Two different EGR1 guide RNA sequence and Cas9 were encoded in V2 CRISPR-GFP or Cherry plasmid (one for each guide). Guide sequences were designed in the first exon of EGR1 with CRISPOR software (http://crispor.tefor.net/) and cloned in one of the two lentiviral vectors : EGR1 guide 1 F : 5'-AAACGGCCGGGTTACATGCGGGGC-3'; R : 5'-CACCGCCCCGCATGTAACCCGGCC-3', EGR1 guide 2 F : 5'-AAACTCGGCGTAGGCCACTGCTTAC-3'; R : 5'-CACCGTAAGCAGTGGCCTACGCCGA-3'. For lentiviral production, 293T were co-transfected with plasmid of interest along with pCMV and pMD2.G plasmids using jetPRime reagent (Polyplus transfection, Ozyme) according to the manufacturer's instructions. Retroviral particules were collected 48h after transfection and concentrated by ultracentrifugation. THP-1 cells were co-transduced with a MOI of 2.5 with both guides and single-cell sorted based on their positive GFP and Cherry expression (BD Influx). EGR1 knockout was assessed by PCR and Sanger sequencing with the following primers : F : 5'-ATAGAGGCGGATCCGGGGAGTC-3'; R : 5'-GAAACCCGGCTCTCATTCTAAGATC-3'. 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 monoclonal anti-CSF-1R antibody (sc-46662, 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.