Antigen

Antigen Class

Unclassified

Antigen

Unclassified

Cell type

Cell type Class

Epidermis

Cell type

Epidermis

MeSH Description

The external, nonvascular layer of the skin. It is made up, from within outward, of five layers of EPITHELIUM: (1) basal layer (stratum basale epidermidis); (2) spinous layer (stratum spinosum epidermidis); (3) granular layer (stratum granulosum epidermidis); (4) clear layer (stratum lucidum epidermidis); and (5) horny layer (stratum corneum epidermidis).

Sample

source_name

epidermis

condition

TNFa

time

2h

chip_antibody

H3K27Ac

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

for ChIP-seq: Purified migratory LCs were fixed with 1% formaldehyde for 15 min and the reaction quenched with 0.125 M 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 100–200 bp (Covaris). Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat to remove crosslinks, 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. Genomic DNA regions of interest were isolated using appropriate antibodies. Complexes were washed, eluted from the beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation (at 37 °C for 1 h, followed by 4 h at 65 °C) overnight at 65 °C, and ChIP DNA was purified by phenol–chloroform extraction and ethanol precipitation. For RNA-seq: RNA was isolated using RNeasy mini kit (Qiagen) as per the manufacturer's protocol. RNA concentration and integrity was determined with an Agilent Bioanalyser (Agilent Technologies, Santa Clara, CA). All the samples had a RNA integrity number of 7.0 or above and were taken forward for labelling. for Chip-seq: 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 using TruplexTM –FD prep kit (Rubicon Genomics, USA). After a final PCR amplification step, the resulting DNA libraries were quantified using Qubit (Life technologies, USA) and 75 nucleotide single-end reads were sequenced Illumina HiSeq2500 in the DNA sequencing core of the Cincinnati Children's Hospital Medical Center. For RNA-seq: RNA-seq libraries were generated from 300 ng total RNA with an RNA Sample Prep Kit (Illumina) according to a standard protocol. The libraries were sequenced with Illumina HiSeq2500 in the DNA sequencing core of the Cincinnati Children's Hospital Medical Center. Each sample was used to generate 2 × 107 reads with 75–base pair paired-end sequencing.

Sequencing Platform

instrument_model

Illumina HiSeq 2500

hg38

Number of total reads

6932623

Reads aligned (%)

93.8

Duplicates removed (%)

26.8

Number of peaks

5422 (qval < 1E-05)

hg19

Number of total reads

6932623

Reads aligned (%)

93.2

Duplicates removed (%)

27.3

Number of peaks

5430 (qval < 1E-05)