title: THP-1 EGR1 CRISPR#3/clone 21 CFMS
organism: Homo sapiens
description: Protocols: 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.