GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM2295947 Query DataSets for GSM2295947
Status Public on Aug 29, 2016
Title PolII_ChIPSeq
Sample type SRA
Source name B cell
Organism Homo sapiens
Characteristics cell line: BJAB B-cell line
cell type: Cell line derived from Burkitt's lymphoma
passage: Multiple
chip antibody: 8WG16
chip antibody vendor: Covance
chip antibody catalog #: MMS-126R
chip antibody lot #: Not Available
Extracted molecule genomic DNA
Extraction protocol Approximately 30 to 50 million cells were cross-linked in 1% formaldehyde for 5 mins and quenched with 1 M Tris pH 8.0. Cells were washed twice with cold PBS and placed in 1 ml Low Salt Buffer (150 mM NaCl, 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.5% NP-40, 1% Triton X-100) with complete protease inhibitor mixture (Roche Applied Science). Cells were sonicated for 15 mins (20 sec. on, 40 sec. off) with a Sonicator Ultrasonic Processor XL (Misonix Incorporated). Lysate was spun down at 13200 rpm for 20 mins and immunoprecipitation performed with 10 µg 8WG16 (Covance, MMS-126R), normal mouse IgG (Santa Cruz, sc-2025), normal rabbit IgG (Santa Cruz, sc-2027), SPT5 (Santa Cruz sc-28878), TRIM28/TIF1b (Abcam ab622553), OGA/NCOAT (Santa Cruz sc-376429), or O-GlcNAc (RL2; Santa Cruz sc-59624) overnight at 4°C. 50 µl protein G beads (Roche), pre-blocked with 0.5% BSA, and were incubated with the lysate for 3 hrs at 4°C. Beads were washed for 5 mins 4 times with RIPA Buffer (10 mM Tris, pH 7.6; 150 mM NaCl; 1 mM EDTA; 1% Triton X-100; 0.1% Sodium deoxycholate; 0.1% SDS) and once with Rinse Buffer (10 mM Tris, pH 7.6; 50 mM NaCl; 1 mM EDTA). The cross-link is reversed in 100 µl Elution Buffer 1 (10 mM Tris, pH 7.6; 1 mM EDTA; 1% SDS) for 10 min at 65°C. 150 µl Elution Buffer 2 (10 mM Tris, pH 7.6; 1 mM EDTA; 0.67% SDS) is added and treated with RNase A (Roche Applied Science) for 30 min at 37°C, and then with Proteinase K (Roche Applied Science) overnight at 65°C. DNA is recovered using a MinElute PCR Purification Kit (QIAGEN). DNA was quantified by Qubit dsDNA High Sensitivity Quantification. Libraries were constructed with 25 ng DNA using the KAPA Hyper Prep Kit (KAPA Biosystems) with NEXTflex DNA Barcodes (BIOO Scientific).
Libraries were constructed with 25 ng DNA using the KAPA Hyper Prep Kit (KAPA Biosystems) with NEXTflex DNA Barcodes (BIOO Scientific).
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
Data processing RTA 2.4.6 Illumina instrument run time analysis software
Bcl2fastq 2.17 Barcode demultiplexed allowing 1 mismatch
Trimmomatic 0.30 with options SE -­threads 16 -­phred33 ILLUMINACLIP: adapters.fa:2:36:10 LEADING:10 TRAILING:10 MAXINFO:50:0.97 MINLEN:20
Bowtie2 2.1.0 with options –p 8 –x bowtie2_ref/genome_prefix –U read1.fastq –S result.sam
macs 2.1.0 with default options except -B for the signal tracks
Genome_build: hg19
Submission date Aug 28, 2016
Last update date May 15, 2019
Contact name Brian Andrew Lewis
Phone 301-435-8323
Fax 301-496-9956
Organization name National Cancer Institute-NIH
Department metabolism Branch
Lab Lewis Lab
Street address 9000 Rockville Pike
City Bethesda
State/province MD
ZIP/Postal code 20892
Country USA
Platform ID GPL18573
Series (1)
GSE86154 O-GlcNAc Aminidase is an RNA Pol II Elongation Factor Coupled to Pausing Factors SPT5 and TIF1b
SRA SRX2059874
BioSample SAMN05713073

Supplementary file Size Download File type/resource
GSM2295947_un_2_peaks.narrowPeak.gz 543.9 Kb (ftp)(http) NARROWPEAK
GSM2295947_un_2_treat_pileup.bedGraph.gz 775.1 Mb (ftp)(http) BEDGRAPH
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap