title: undiff_d0_r1
description: Protocols: The mouse Brie knockout CRISPR pooled library (Doench et al 2016) on the lentiGuide-Puro backbone was transduced into cas9-expressing conditionally-immortalized macrophage progenitors. Brie library transduced progenitors were differentiated into macrophages by removal of beta-estradiol and addition of MCSF. Cells were differentiated for seven days before harvest. Library samples were taken on day 0 of differentiation, and day 7 of differentiation. In addition, a sample of progenitors was not differentiated and passaged in parallel for 7 days. Three independent experiments were performed. Cells were lysed in 50mM Tris, 50mM EDTA, 1% SDS, pH 8 + 100ug/ml proteinase K, RNAse treated, then incubated with ammonium acetate for a final concentration of 1.875M. After centrifugation, supernatants were incubated with isopropanol to precipitate genomic DNA. After centrifugation gDNA pellets were washed in 70% ethanol. After a final centrifugation, gDNA pellets were air dried and resuspended in 10mM Tris HCL pH 8.5 + 0.1mM EDTA.  Library was constructed as previously described, using primers for the Brie library (Doench et al., 2016). Briefly the sgRNA loci were amplified by PCR using Illumina compatible primers that allow for sample multiplexing. Amplicons were purified then pooled.
attributes.tag: ENA first public
attributes.value: 2026-04-29
attributes.tag: INSDC center name
attributes.value: UC Berkeley
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-15740:undiff_d0_r1
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: cas9-expressing, HoxB8 conditionally-immortalized macrophages
attributes.tag: cell type
attributes.value: macrophages
attributes.tag: collection date
attributes.value: not collected
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: mouse postnatal
attributes.tag: disease
attributes.value: normal
attributes.tag: genetic modification
attributes.value: genome-wide CRISPR knockout library: Brie (Doench et al 2016) in lentiGuide-puro
attributes.tag: genotype
attributes.value: cas9-expressing C57BL/6
attributes.tag: geographic location (country and/or sea)
attributes.value: not collected
attributes.tag: isolate
attributes.value: not applicable
attributes.tag: organism part
attributes.value: bone marrow
attributes.tag: replicate
attributes.value: 1
attributes.tag: sample name
attributes.value: E-MTAB-15740:undiff_d0_r1
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: stimulus
attributes.value: undifferentiated