|
|
GEO help: Mouse over screen elements for information. |
|
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 Selector |
Raw data are available in SRA |
|
|
|
|
|