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| Status |
Public on Aug 29, 2016 |
| Title |
PolII_ChIPSeq |
| Sample type |
SRA |
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| Source name |
B cell
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| 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
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| 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).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
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| 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
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| Submission date |
Aug 28, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Brian Andrew Lewis |
| E-mail(s) |
lewisbri@mail.nih.gov
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| Phone |
301-435-8323
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| Fax |
301-496-9956
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| Organization name |
National Cancer Institute-NIH
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| Department |
metabolism Branch
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| Lab |
Lewis Lab
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| Street address |
9000 Rockville Pike
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL18573 |
| Series (1) |
| GSE86154 |
O-GlcNAc Aminidase is an RNA Pol II Elongation Factor Coupled to Pausing Factors SPT5 and TIF1b |
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| Relations |
| 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 Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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