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Sample GSM4928635

Query DataSets for GSM4928635

Status

Public on Nov 22, 2020

Title

LuCaP_173.2_INPUT

Sample type

SRA

Source name

Prostate cancer patient-derived xenograft (PDX)

Organism

Homo sapiens

Characteristics

histology: Prostate adenocarcinoma
chip antibody: Input control

Extracted molecule

genomic DNA

Extraction protocol

Frozen tissue (20-30mg for histone mark ChIP and 50-80mg for transcription factor ChIP) was pulverized using the CryoPREP dry impactor system (Covaris). The tissue was then fixed using 1% formaldehyde (Thermo fisher) in PBS for 18 minutes either at 37 degrees Celsius (histone mark ChIP) or at room temperature (transcription factor ChIP) and was quenched with 125 mM glycine. Chromatin was lysed in ice-cold lysis buffer (50mM Tris, 10mM EDTA, 1% SDS with protease inhibitor for histone mark ChIP; 0.1% SDS, 0.5% sodium deoxycholate and 1% NP-40 with protease inhibitor for transcription factor ChIP) and was sheared to 300-800 bp using the Covaris E220 sonicator (105 watt peak incident power, 5% duty cycle, 200 cycles/burst for 10 minutes for histone mark ChIP; 140 watt peak incident power, 5% duty cycle, 200 cycles/burst for 20 minutes for transcription factor ChIP). Five volumes of dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris HCl pH 8.1) were added to chromatin for histone mark ChIP. The sample was then incubated with antibodies (H3K27ac, Diagenode, C15410196; H3K27me3, Cell Signaling 9733S; H3K4me3, Diagenode C15410003 premium; FOXA1, ab23738, Abcam) coupled with protein A and protein G beads (Life Technologies) at 4 degrees Celsius overnight. The chromatin was washed with RIPA wash buffer (100 mM Tris pH 7.5, 500 mM LiCl, 1% NP-40, 1% sodium deoxycholate) for 10 minutes six times and rinsed with TE buffer (pH 8.0) once. DNA was purified using MinElute column followed by incubation in the de-crosslinking buffer (1% SDS, 0.1M NaHCO3 with Proteinase K and RNase A) at 65 degrees Celsius.
DNA sequencing libraries were prepared using the ThruPLEX-FD Prep kit (Rubicon Genomics). Libraries were sequenced using 150-base paired end reads on an Illumina platform (Novogene).

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Description

LuCaP_173.2_INPUT

Data processing

Illumina Casava1.7 software used for basecalling.
Reads were aligned to hg19 using BWA.
MACS2 was used for peak calling.
Genome_build: hg19
Supplementary_files_format_and_content: bigwig files represent normalized read counts in treatment conditions, bed files contain coordinates for MACS2 peak calls

Submission date

Nov 21, 2020

Last update date

Nov 22, 2020

Contact name

Sylvan Baca

Organization name

Dana–Farber Cancer Institute

Street address

450 Brookline Ave

City

Boston