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Sample GSM7823548 Query DataSets for GSM7823548
Status Public on Oct 06, 2023
Title 311E cell line 9nM ABBV075 96hr IgG rep 2 Experiment 2
Sample type SRA
 
Source name 311E cell line
Organism Mus musculus
Characteristics tissue: 311E cell line
cell line: Genetically engineered mouse model of NUT Carcinoma
genotype: Brd4-NUTM7
cut&run antibody: IgG (EpiCypher, 13-0042K)
treatment: ABBV-075, 9nM, 96hr
Growth protocol DMEM, 10% fetal bovine serum, 1x glutamax, 1% penicillin/streptomycin. Cells were cultured at 37C at 5% CO2.
Extracted molecule genomic DNA
Extraction protocol Cells were fixed with 0.1% formaldehyde for 1 minute. Tissue was fixed with 0.2% formaldehyde for 2 minutes. Cells were bound to Con-A beads and incubated overnight with target antibodies. Bead bound cells were washed and incubated with pAG-MNase for 60 minutes at 4degC before being washed and incubated with CaCl2 to activate MNase. Reaction was stopped with EGTA,EDTA containing buffer and incubated for 10minutes at 37degC to release DNA. SDS and proteinase K were added to each sample before overnight incubation at 55degC to reverse cross link. DNA was purified using oligo clean and concentrator. Yields were determined using Qubit 2.0 Fluorometer with 1x dsDNA HS assay kit
Immunoprecipitated DNA was quantified with Qubit 2.0 DNA HS Assay (ThermoFisher, Massachusetts, USA) and quality was assessed by Tapestation genomic DNA Assay (Agilent Technologies, California, USA). Library preparation was performed using KAPA Hyper Prep kit with PCR (Roche, Basel, Switzerland) per manufacturer’s recommendations. Library quality and quantity were assessed with Qubit 2.0 DNA HS Assay (ThermoFisher, Massachusetts, USA), Tapestation High Sensitivity D1000 Assay (Agilent Technologies, California, USA), and QuantStudio ® 5 System (Applied systems, California, USA). Illumina® 8-nt unique dual-indices were used. Equimolar pooling of libraries was performed based on QC values and sequenced on an Illumina® NovaSeq X Plus 10B (Illumina, California, USA) with a read length configuration of 150 PE for 8M PE reads (4M in each direction).
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NovaSeq 6000
 
Description 23122FL-07-02-02
Data processing library strategy: CUT&RUN
Reads were trimmed for adaptor sequences using trimmomatic v0.36 and then aligned to concatenated mm39/Genome Reference Consortium mouse Reference 39 (GRCm39) assembly of the human genome, using bowtie2 v2.3.1 with options --local --very-sensitive-local --no-unal --nomixed --no-discordant --phred33. Only reads with a MAPQ ≥ 30 were retained using samtools v1.3.1. PCR duplicates were removed with picard MarkDuplicates v2.9.2. Coverage-based tracks were generated with deepTools bamCoverage with options --binSize 1 -normalizingUsing RPGC --effectiveGenomeSize 2800000000 -extendReads --samFlagInclude 64. CUT&RUN profiles were then calibrated by spike-in normalization using the E. Coli DNA as the internal reference using a similar method as that done for ChIP-seq[25]. Specifically, the Occupancy Ratio (OR) was calculated as the product of IgG spike-in reads and immunocleaved (IC) human reads, divided by the product of IgG human reads and IC spike-in reads. Spike-in normalized tracks were generated with deepTools bamCoverage with options --binSize 1 --normalizeUsing RPGC --effectiveGenomeSize 2800000000 --extendReads --samFlagInclude 64 --scaleFactor OR.epic2 v0.0.52 was used for domain calling on the canonical human chromosomes with options --bin-size 200 --gaps-allowed 5 with the corresponding IgG library set as the control. Called domains were filtered by signal strength and by domain size using the ROSE algorithm (Rank Ordering of Super-Enhancers)[26]. Domains that exceeded both the signal cutoff and the size threshold were retained. We frequently observed closely spaced filtered domains that corresponded to larger domains based on CUT&RUN signal tracks. To span and capture these regions filtered domains within 25 kb of one another were combined. A high confidence list of “megadomains” was created by merging filtered domains identified in both logical replicates.
Assembly: GRCm39/mm39
 
Submission date Oct 04, 2023
Last update date Oct 06, 2023
Contact name Chris French
E-mail(s) cfrencH@bwh.harvard.edu
Phone 617-525-4415
Organization name Brigham and Women's Hospital
Department Pathology
Lab French
Street address 77 avenue louis Pasteur, NRB 652
City Boston
State/province MA
ZIP/Postal code 02115
Country USA
 
Platform ID GPL24247
Series (2)
GSE241476 A genetically engineered mouse model of NUT carcinoma (CUT&RUN)
GSE241477 A genetically engineered mouse model of NUT carcinoma
Relations
BioSample SAMN37686732
SRA SRX21992291

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA

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