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Sample GSM4995479 Query DataSets for GSM4995479
Status Public on Dec 31, 2020
Title Infected SP-R210L(DN) Cells PU.1 ChIP 1
Sample type SRA
Source name Modified RAW264.7 RAW Cell Line
Organism Mus musculus
Characteristics cell line background: RAW264.7 RAW
sp-r210 isoform expression: RAW264.7 cells with SP-R210ct pTriex-2-Neo vector
iav infection: PR8 IAV Infected, MOI 1
chip antibody: anti-PU.1 antibody-bound Goat-anti-rabbit IgG Dynabeads
chip antibody source: Invitrogen Cat #MA5-15064, clone E.388.3
Treatment protocol Infected cells were infected with MOI 1 of PR8 influenza A virus for 2 hours in infection media (1:1 DMEM/PBS), after which virus-containing media was removed and replaced with DMEM with 10% FBS. Infection was allowed to porceed for 22 hour for a total 24 hour infection period. Control cells had media replaced with infection media for 2 hours, after which 10% FBS containing DMEM was replaced for the remainder of the time.
Growth protocol RAW264.7 cells were stably transfected with empty for SP-R210ct pTriex-2-Neo vectors and selected using 600ug/mL neomycin sulfate to generate WT and SP-R210L(DN) RAW264.7 cells, respectively. Five plates of WT and SP-R210L(DN) RAW 264.7 macrophages were cultured at density of 1E7 for each sample.
Extracted molecule genomic DNA
Extraction protocol Cells were removed from plaates and cross-linked with 1% formaldehyde for 10 minutes. Nuclei were extracted from cell pellets via 10 minute incubation via incubation in L1 lysis buffer (50mM Hepes KOH, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, pH7.5) and L2 lysis buffer (200 mM NaCl, 1mM EDTA, 0.5mM EGTA, 10mM Tris, pH8.0). Pelleted nuclei was resuspended in 5 mL of L3 buffer (1mM EDTA, 0.5mM EGTA, 10mM Tris, 1x protease inhibitor) and DNA from nuclei was fragmented using the Bioruptor sonicator for 30 min (30s pulses, 30s pauses in between for 10 min, run 3 times). 25 uL of sheared DNA was aliquoted to run as input DNA. anti-PU.1 antibody , anti-H3K4me3, anti-H3K9me3 , or anti-H3K27me3 bound Goat-anti-rabbit IgG Dynabeads were incubated with 500 uL chromatin in TE buffer (with 0.1% deoxycholate, 1% Triton X-100) overnight . Protein/DNA complexes were washed and captured with a Magnetic Particle Concentrator. DNA-protein crosslink was reversed via incubation at 65oC overnight and then treated with 1 mg/mL proteinase K for 2 hours at 37oC. DNA was extracted using phenol and chloroform extraction and precipitated using 100% EtOH. The dried DNA pellet was reconstituted in 50 uL H2O treated with 330ug/mL of RNase A for 2 hours at 37oC and then recovered using the QIAquick PCR Purification kit
ChIP-seq libraries were created using Kapa HyperPrep Kit (Roche Sequencing and Life Science). The unique dual index sequences (NEXTFLEX® Unique Dual Index Barcodes, BioO Scientific) were incorporated in the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent Technologies).
Pooled libraries were diluted and then denatured using the Illumina protocol. The denatured libraries were loaded onto a TruSeq v2 Rapid flow cell on an Illumina HiSeq 2500 and run for 50 cycles using a single-read recipe.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2500
Description DNInf_ChIP_PU1_1
Data processing FASTQ sequences were aligned to the mm10 genome using the mem function of the bwa package. Peaks were identified from the bed files using the callpeaks function of MACS2 (v2.1.0). Peak annotation and identification of overlapping peaks was done via ChIPpeakAnno (v3.14) using the UCSC mm10 annotated genome.
Genome_build: mm10
Supplementary_files_format_and_content: narrowPeak files are provided for each respective sample
Submission date Dec 30, 2020
Last update date Jan 01, 2021
Contact name Zissis Chroneos
Organization name Penn State College of Medicine
Department Pediatrics
Street address 500 University Drive
City Hershey
State/province PA
ZIP/Postal code 17036
Country USA
Platform ID GPL17021
Series (2)
GSE164034 Genomic and epigenomic adaptation in SP-R210 (Myo18A) isoform-deficient macrophages [ChIP-seq]
GSE164036 Genomic and epigenomic adaptation in SP-R210 (Myo18A) isoform-deficient macrophages
BioSample SAMN17184594
SRA SRX9756287

Supplementary file Size Download File type/resource
GSM4995479_DN_PU1ChIP_inf1_peaks.narrowPeak.gz 99.6 Kb (ftp)(http) NARROWPEAK
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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