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Sample GSM4337384 Query DataSets for GSM4337384
Status Public on Sep 02, 2021
Title TS060911A_X_FAIRE-seq
Sample type SRA
 
Source name CD19+ B cells
Organism Homo sapiens
Characteristics tissue: tonsil
disease state: healthy
antibody: --
Extracted molecule genomic DNA
Extraction protocol Cells were formaldehyde cross-linked (1% final concentration) for 10 minutes in RPMI with 10% FBS. Quenching was performed by addition of 1M glycine (125 mM final concentration) with gentle mixing for 10 minutes at room temperature. Chromatin was lysed for 10 minutes sequentially in L1 buffer (50mM Hepes KOH pH 7.5, 140mM NaCl, 1mM EDTA pH 8.0, 10% glycerol, 5% NP40 and 0.25% Triton X-100) followed by L2 buffer (200mM NaCl, 1mM EDTA pH 8.0, 0.5mM EGTA pH 8.0, 1M and Tris pH 8.0). The pellets were resuspended in 300 ul of buffer L3 (1mM EDTA pH 8.0, 0.5mM EGTA pH 8.0, 1M Tris pH 8.0, 100mM NaCl, 0.1% Na-deoxycholate and 5mg/ml N-lauroyl sarcosine) and sonicated to an average size of 500bp using a Diagenode Bioruptor. The soluble DNA fraction was isolated by performing two consecutive phenol:chloroform:isoamylalcohol (25:24:1, pH 8) extractions followed by a chloroform-isoamyl alcohol (24:1) extraction. Precipitated DNA was resuspended in 10mM Tris-HCl (pH 7.4) and incubated with 10 µg of RNase A for 1hr. DNA was purified with QIAquick PCR purification columns.
Cells were formaldehyde cross-linked (1% final concentration) for 10 minutes in RPMI with 10% FBS. Quenching was performed by addition of 1M glycine (125 mM final concentration) with gentle mixing for 10 minutes at room temperature. Cells were washed with cold PBS and lysed in an SDS buffer (1% SDS, 10 mM EDTA, 50 mM Tris pH 8.0) with protease inhibitors before chromatin was fragmented using a Diagenode Bioruptor to an average size of less than 300 bp as determined using an Agilent Bioanalyzer 2100. Lysates were immunoprecipitated with the listed antibodies conjugated to Protein-A Dynabeads (5 ug antibody incubated with beads for 2-4 hours at 4°C) overnight at 4°C. The beads were then washed with the following buffers each for 3 minutes at 4°C - low salt buffer (150 mM NaCl, 50 mM Tris pH 8.0, 0.1% SDS), high salt buffer (500 mM NaCl, 50 mM Tris pH 8.0, 0.1% SDS), LiCl buffer (250 mM LiCl, 50 mM Tris pH 8.0, 0.5% Na deoxycholate, 1% NP40), and TE buffer (10 mM Tris pH 8.0, 1 mM EDTA). After washing, crosslinks were reversed overnight at 65°C in 250 uL elution buffer (1% SDS, 100 mM NaHCO3) followed by addition of proteinaseK and incubation at 56°C for 1 hour. DNA was purified with QIAquick PCR purification columns.
At least 3 ng of DNA was used to for indexed library preparation using Illumina TruSeq adapters and standard Illumina protocols. Fragments were size selected (average fragment-size: 200-400 bp for ChIP; 50-200 for FAIRE) using Agencourt AMPure XP beads prior to amplification. Samples were pooled (6-10 per lane) and sequenced.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2000
 
Data processing Reads were aligned to hg19 with bowtie2 (v2.2.5) using default settings.
Reads in ENCODE blacklisted regions were removed with samtools (v1.9).
Peaks were called with MACS2 (v2.1.0.20150420) with the parameters --nomodel --shiftsize=150 (ChIP) or --nomodel --shiftsize=50 (FAIRE) and input controls.
DiffBind (v2.14.0) was used to derive consensus peak sets for each histone mark and accessible chromatin, requiring peaks to overlap in at least three samples for each ChIP-seq or FAIRE-seq assay in order to be merged and retained. The consensus peaksets for each assay were then concatenated and merged to derive a list of putative regulatory elements with bedtools2 (v2.29.2).
DiffBind was then used to determine differentially bound/accessible regions between sample groups for each assay. ChIPseeker (v1.22.0) was used to annotate peaks with hg19 UCSC knownGene annotations.
Genome_build: hg19
Supplementary_files_format_and_content: narrowPeak format files containing peaks for each sample as called by MACS2.
 
Submission date Feb 24, 2020
Last update date Sep 02, 2021
Contact name Jared Andrews
E-mail(s) jared.andrews@stjude.org
Organization name St. Jude Children's Research Hospital
Department Developmental Neurobiology
Street address 262 Danny Thomas Pl
City Memphis
State/province Tennessee
ZIP/Postal code 38105
Country USA
 
Platform ID GPL11154
Series (2)
GSE145841 Key Super Enhancers Drive Tumor-Suppressing Transcription Feedback Programs in Mature B Cell Cancers (ChIP-seq, FAIRE-seq)
GSE145848 Key Super Enhancers Drive Tumor-Suppressing Transcription Feedback Programs in Mature B Cell Cancers
Relations
BioSample SAMN14175916
SRA SRX7795933

Supplementary file Size Download File type/resource
GSM4337384_TS060911A_X.narrowPeak.gz 580.7 Kb (ftp)(http) NARROWPEAK
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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