Antigen

Antigen Class

TFs and others

Antigen

CTCF

Cell type

Cell type Class

Blood

Cell type

Erythroid Cells

MeSH Description

The series of cells in the red blood cell lineage at various stages of differentiation.

Sample

source_name

iPSC diffrentiation to Erythroid cells

cell type

Erythroid

antibody

CTCF antibody 07-729 (Merck Millipore)

differentiation day

Day 21

genotype

Wild type

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

isolation protocol: Cells were fixed at 2.5x10^6 cells/ml of 10% FCS RPMI media (Gibco) with 1% formaldehyde (SIGMA) for 10min at RT. Fixed cells were lysed at 5x10^7 cells/ml of cell lysis buffer (5mM PIPES (Gibco), 85mM KCl, 0.5% NP-40 (SIGMA)) + proteinase inhibitor (PI) (ROCHE) on ice for 20min. The lysed fixed nuclei were lysed in nuclear lysis buffer (50mM Tris-HCl, 10mM EDTA (both from Life Technologies) 1% SDS (SIGMA)) +PI at a concentration of 1x10^8 cells/ml. The nuclear lysis was sonicated using the Covaris S220 Focused-ultrasonicator in 130μl microTUBEs (Covaris). The sonicated lysate was spun at max speed at 10°C to remove insoluble material. The clear fixed chromatin was diluted 1 in 10 with RIPA buffer w/o SDS +PI (10mM Tris-HCl, 1mM EDTA, 0.5mM EGTA (SIGMA), 1% Triton X-100 (SIGMA), 0.1% Na Deoxycholate (SIGMA), 140mM NaCl). The diluted chromatin was precleared twice with 10µl of a 1:1 mix of Protein A and Protein G Dynabeads (Invitrogen) by incubating at 4°C for 30mins and discarding the beads. The pre-cleared chromatin was incubated with the appropriate amount of antibody coupled to a 100µl 1:1 mix of Protein A and Protein G Dynabeads at 4°C overnight. The antibodies used are: RNA Polymerase II N-20 antibody SC-899 (Santa Cruz Biotechnology) 6μg per 1ml of chromatin from 1x10^7 cells; Monoclonal anti-acetyl-Histone H3 (Lys27) antibody 17-683 (Merck Millipore) 2μg per 1ml of chromatin from 1x10^7 cells; Polyclonal anti-trimethyl-Histone H3 (Lys4) antibody 07-473 (Merck Millipore) 1μg per 1ml of chromatin from 1x10^7 cells; Polyclonal anti-monomethyl-Histone H3 (Lys4) antibody ab195391 (Abcam) 4μg per 1ml of chromatin from 1x10^7 cells; Polyclonal anti-GATA1 antibody ab11852 (Abcam) 8μg per 1ml of chromatin from 1x10^7 cells; Policlonal anti-CTCF antibody 07-729 (Merck Millipore) 10μl serum per 1ml of chromatin from 1x10^7 cells. Polyclonal anti-KLF1 antibody was kindly provided by the Perkins Laboratory, Translational Research Institute, Brisbane, Australia (used 30μl serum per 1ml of chromatin from 1x10^7 cells). Chromatin bound beads were washed twice with RIPA (10mM Tris-HCl, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na Deoxycholate, 140mM NaCl) +PI, then twice with high salt RIPA (same as RIPA but with 500mM NaCl) +PI, once with LiCl RIPA (same as RIPA but with 250mM LiCl instead of NaCl) +PI and twice with TE buffer. Chromatin was eluted off beads using 20mM Tris-HCl, 5mM EDTA, 50mM NaCl, 1% SDS, 130µg/ml RNase A, 65µg/ml Proteinase K. Eluted chromatin and Input samples were incubated at 65°C overnight to de-crosslink DNA from protein. DNA was purified by phenol-chloroform extraction and ethanol precipitation with NaOAc, and 1 µl GlycoBlue (Invitrogen). ChIP enrichment was determined by RT-qPCR NEBNext Ultra II DNA Library Prep kit for Illumina (NEB).

Sequencing Platform

instrument_model

NextSeq 500

hg38

Number of total reads

23672626

Reads aligned (%)

93.9

Duplicates removed (%)

8.7

Number of peaks

38953 (qval < 1E-05)

hg19

Number of total reads

23672626

Reads aligned (%)

93.2

Duplicates removed (%)

8.9

Number of peaks

38789 (qval < 1E-05)