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Sample GSM2295953

Query DataSets for GSM2295953

Status

Public on Aug 29, 2016

Title

Rabbit_IgG_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: rabbit IgG
chip antibody vendor: Santa Cruz
chip antibody catalog #: sc-2027
chip antibody lot #: J2014

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

E-mail(s)

lewisbri@mail.nih.gov

Phone

301-435-8323

Fax

301-496-9956