Antigen

Antigen Class

TFs and others

Antigen

CTCF

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

epidermal keratinocytes from foreskin

cell type

epidermal keratinocytes

chip antibody

CTCF, Diagenode C15410210, lot A2359-00234D

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

When keratinocytes are under pre-confluency, they were cross-linked by 1% paraformaldehyde for 8 min for H3K27ac and for 15 min for MED1/CTCF and quenched with 0.125M glycine. Whole cell lysates (for H3K27ac) or purified chromatin (MED1/CTCF) were sonicated by Covaris S2200 ultrasonicator (Covaris, Inc.). The shearing condition was optimized to obtain the DNA to an average length of 300±50 bp, in which size of DNA was verified by Agilent 2100 Bioanalyzer (Agilent Technologies). Sheared chromatin was immunoprecipitated with Protein A-coated magnetic beads (Diagenode) preincubated with antibodies: 3 ug of antibody against H3K27ac (ab4729, Abcam), 1 ug CTCF antibody (Diagenode) and 4.5 ug MED1 antibody (Bethyl). Experiments were carried out in replicates (2-3), and a chromatin input sample was used as a reference to subtract background for peak calling. Complexes were washed, eluted from the beads with washing buffer and crosslinks were reversed by incubation at 65C for 4 hr. Immunoprecipitated DNA along with genomic DNA (Input) were purified using IPure v2 kit (Diagenode). IP efficiency was confirmed by qPCR using primers provided in the kits. DNA Sequencing libraries were generated using the Accel-NGS 2S Plus DNA library kits (Swift Sciences) and amplified by PCR for 11 cycles. To remove high MW smear in the library, right side size selection was conducted by using SPRI beads (Beckman Coulter). The library was quantified by Agilent 2100 Bioanalyzer with the High sensitivity DNA assay. They were sequenced on an Illumina HiSeq 4000 (UCSF Center of advanced technology) in a single strand 50bp run (combining 8 libraries per lane).

Sequencing Platform

instrument_model

Illumina HiSeq 4000

hg38

Number of total reads

48109548

Reads aligned (%)

98.2

Duplicates removed (%)

11.9

Number of peaks

59035 (qval < 1E-05)

hg19

Number of total reads

48109548

Reads aligned (%)

97.7

Duplicates removed (%)

12.6

Number of peaks

58853 (qval < 1E-05)