Antigen

Antigen Class

Unclassified

Antigen

Unclassified

Cell type

Cell type Class

Epidermis

Cell type

Keratinocytes

MeSH Description

Epidermal cells which synthesize keratin and undergo characteristic changes as they move upward from the basal layers of the epidermis to the cornified (horny) layer of the skin. Successive stages of differentiation of the keratinocytes forming the epidermal layers are basal cell, spinous or prickle cell, and the granular cell.

Sample

source_name

Human primary keratinocytes

cell type

Human primary keratinocytes

input

Y_INPUT2

barcode

CACGATTC

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

For H3K27ac: Approximately 4x106 cells were cross-linked with 10% formaldehyde at room temperature for 15 min. After quenching with 125mM glycine for 6min, cells were washed in ice-cold PBS (plus Protease Inhibitor (PI) Cocktail tablet (Sigma) and 100mM PMSF) and collected by scraping in Nuclei EZ lysis buffer (Sigma, plus PI and 100mM PMSF). Nuclei were collected by centrifugation (500xg for 5min at 4°C). In order to increase nuclei collection, pellets were further re-suspended in a Hypotonic Buffer (10mM HEPES, 10mM KCl, 0.1mM EDTA, 0.1mM EGTA, pH8.0) for 10min in ice followed by NP40 (0.5% v/v) and 20sec vortex at maximum speed. Nuclei were collected by centrifugation (14.000rpm, 4°C, 30sec), washed with Wash Buffer C (with PI) (truChIP Chromatin Shearing Kit, Covaris, Brighton, UK) and re-suspended in Shearing buffer D3 (plus PI and 100mM PMSF) (truChIP Chromatin Shearing Kit, Covaris). Nuclei were vortexed for 3 rounds of 10sec and left 15min in ice before freezing in -80°C for later use. After thawing, 130μl nuclei suspensions were transferred into microTUBE (Covaris) and sonicated using a Covaris E220 (10min: duty cycle 2%, Intensity 5, Cycles per Burst 200). Samples were collected into 1.5ml tubes, centrifuged at 10000xg for 10min at 4°C and supernatants transferred into 2ml tubes. DNA concentrations were calculated after DNA isolation using Qiaquick PCR (Qiagen) using Qubit Nucleic Acid Quantification (Invitrogen). For ChIP a modified protocol of the Magna ChIP A/G (Merck #17-10085) protocol was used. In brief 5μg of mice anti-H3K27ac Ab or IgG were coupled with Protein A/G beads at 4°C for 4h. Coupled Ab-beads were mixed with 5μg of samples and incubated overnight at 4°C with rotation. Ab-beads were washed as per manufacturer's instructions and protein/DNA complexes eluted in Elution buffer (50mM NaCl and 1%SDS in dH2O) for 4h at 65°C. Reverse cross-linking was performed by adding RNAse for 30min at 37°C and 5mM EDTA, 20mM Tris pH7.5 and 100μg/ml Proteinase for another 1h at 45°C. Supernatants were separated and DNA extracted using the MiniElute PCR purification kit (Qiagen).For BRD4: Frozen cells (9x106) were thawed and fixed with 1% formaldehyde for 15min and quenched with 0.125M glycine. Chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. Extrapolation to the original chromatin volume allowed quantitation of the total chromatin yield. An aliquot of chromatin (30μg) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4μg of antibody against BRD4. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65°C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Illumina sequencing libraries were prepared from the ChIP and Input DNAs by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on Illumina's NextSeq 500 (75nt reads, single end).

Sequencing Platform

instrument_model

NextSeq 500

hg38

Number of total reads

38754692

Reads aligned (%)

98.1

Duplicates removed (%)

1.9

Number of peaks

22887 (qval < 1E-05)

hg19

Number of total reads

38754692

Reads aligned (%)

97.3

Duplicates removed (%)

2.0

Number of peaks

22371 (qval < 1E-05)