Antigen

Antigen Class

Input control

Antigen

Input control

Cell type

Cell type Class

Others

Cell type

Head and Neck Neoplasms

MeSH Description

Soft tissue tumors or cancer arising from the mucosal surfaces of the LIP; oral cavity; PHARYNX; LARYNX; and cervical esophagus. Other sites included are the NOSE and PARANASAL SINUSES; SALIVARY GLANDS; THYROID GLAND and PARATHYROID GLANDS; and MELANOMA and non-melanoma skin cancers of the head and neck. (from Holland et al., Cancer Medicine, 4th ed, p1651)

Sample

source_name

HN120 cis-platin resistant primary HNSCC

cell type

Primary Cisplatin Resistant (PCR) cells

passages

13-15

source

Cisplatin-reistant model

chip antibody

none

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

For chromatin prepration one million cell were cross-linked (10% FBS/DMEM containing 1.5% PFA) for 30 min. Followed by addition of 1/20 volume of 2.5M glycine, next these cells were incubated on ice for 5 min. Crosslinked cellular material was re-dissolved in buffer contacting 10 mM Tris-HCl, pH8, 1 mM EDTA, 0.5 mM EGTA, 1X protease inhibitor cocktail (Roche) followed by sonication for 10-12 cycles (30 sec on/off). Chromatin was pulled by incubation with H3K4me3 or H3K427ac antibody-coupled dynabeads and 150 μl of ChIP buffer (433 mM NaCl, 0.43% sodium deoxycholate, 4.3% Triton X-100). The 1/10 of chromatin mixture was separated for input control. The beads were washed 5x with RIPA buffer and incubated in 100 μl of elution buffer (50 mM Tris-HCl, pH8, 1% SDS, 10 mM EDTA) at 65°C for 20 min with brief vortexing followed by recovery of immunoprecipitated chromatin. The immunoprecipitated chromatin and input samples were de-crosslinked by incubation at 65°C overnight and subsequent incubation with 0.2 μg/μl of RNase A at 37°C for 1 hr and with 0.2 μg/μl of proteinase K at 55°C for 2 hr. The DNA was extracted by phenol/chloroform/isoamylalcohol (49:49:2) and ethanol precipitated in presence of 40 μg of glycogen. After washing with 70% ethanol, pellets were resuspended in 10 mM Tris-HCl, pH8. Multiplexed ChIP-seq libraries were generated using NEBNext ChIP-seq library Prep kit (NEB) following the instructions from the manufacturer. The sequencing was performed on Illumina HiSeq-2500. We employed Dfilter to call ChIP peaks in the naïve, drug-resistant and drug-holiday H3K4me3 and H3K27ac ChIP-seq libraries relative to input controls.

Sequencing Platform

instrument_model

Illumina HiSeq 2500

hg38

Number of total reads

28359395

Reads aligned (%)

97.3

Duplicates removed (%)

10.2

Number of peaks

951 (qval < 1E-05)

hg19

Number of total reads

28359395

Reads aligned (%)

96.6

Duplicates removed (%)

11.6

Number of peaks

790 (qval < 1E-05)