Antigen

Antigen Class

TFs and others

Antigen

Sox11

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

keratinocytes transduced with Sox11-Flag lentivirus

strain background

mixed 129SvEx:C57BL/6

cell type

primary keratinocytes

genotype/variation

Krt14-Cre;Sox11fl/fl;Sox4fl/fl

chip antibody

mouse anti-FLAG antibody (Sigma)

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Nuclei were prepared from approximately 100 million cells and lysed in the sonication buffer (50 mM Tris-HCl pH 8, 10 mM EDTA, and 1% SDS). Chromatin was sheered by sonication in iced water (Bioruptur XL, Diagenode), 30 sec on/30 sec off, for 35 min, to achieve 100–500-bp DNA fragments. After dialysis against the low SDS buffer (20 mM Tris-HCl pH 8, 10 mM EDTA, and 0.01% SDS) overnight, the sonicated samples were centrifuged at 20,000 g for 10 min. The soluble whole-cell extracts were added with Triton X-100 (final 1%) and NaCl (final 150 mM), and incubated with 100 µl of Dynal Protein G magnetic beads that had been pre-incubated with 10-µg mouse anti-FLAG antibody (Sigma) or mouse IgG. Beads were washed 1´ with low-salt immune complex wash buffer (0.1% SDS, 1% TritonX-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH8.1), 1´ with high-salt immune complex wash buffer(0.1% SDS, 1% TritonX-100, 2 mM EDTA, 500 mM NaCl, 20 mM Tris-HCl, pH8.1), 1´ with LiCl immune complex wash buffer (250 mM LiCl, 0.5% NP-40, 0.5% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1), and 1´ with TE containing 50 mM NaCl. Bound complexes were eluted from the beads by heating at 65oC for 30 min (50 mM Tris-Hcl, pH 8.0, 10 mM EDTA, and 1% SDS) in a Thermomix. IgG ChIPs were performed under the same conditions as the experimental ChIPs. Crosslinking was reversed by incubating samples at 65oC for 6 hr. Whole-cell extract DNA reserved from the sonication step was treated in parallel to reverse crosslinking. After crosslink reversal, samples were treated with RNase and proteinase K prior to DNA extraction. Rubicon ThruPlex DNA-Seq library preparation system

Sequencing Platform

instrument_model

Illumina HiSeq 2500

mm10

Number of total reads

68793772

Reads aligned (%)

98.4

Duplicates removed (%)

28.7

Number of peaks

30405 (qval < 1E-05)

mm9

Number of total reads

68793772

Reads aligned (%)

98.2

Duplicates removed (%)

28.7

Number of peaks

30445 (qval < 1E-05)