Antigen

Antigen Class

Input control

Antigen

Input control

Cell type

Cell type Class

Blood

Cell type

G1E-ER4+E2

Tissue

Blood

Lineage

cellLine

Description

Gata1 restored erythroid cells, differentiation induced by estradiol (E2)

Sample

source_name

G1E-ER4 cells

cell type

erythroid

chip ab

none

genotype/variation

wild type G1E-ER4 cells

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

G1E-ER4 cells were crosslinked with 1% formaldehyde in PBS with gentle agitation for 10 minutes at room temperature and then quenched with glycine. Fixed cells were resuspended in 1mL Cell Lysis Buffer (10mM Tris-HCl pH 8, 10mM NaCl, 0.2% NP-40/Igepal) prepared fresh with protease inhibitors (Sigma P8340) and 1mM phenylmethylsulfonyl fluoride (PMSF) and incubated on ice for 10 minutes. Nuclei were pelleted and resuspended in 1mL Nuclear Lysis Buffer (50mM Tris-HCl pH 8, 10mM EDTA pH 8, 1% SDS, prepared fresh with protease inhibitors and PMSF), and incubated on ice for 20 minutes. Samples were then diluted with 0.6mL IP Dilution Buffer (20mM Tris-HCl pH 8, 2mM EDTA pH 8, 150mM NaCl, 1% Triton X-100, 0.01% SDS, prepared fresh with protease inhibitors and PMSF), and sonicated at 4 degrees C (Epishear, Active Motif). After sonication, samples were spun at 4 degrees C to remove debris and added to preclearing reactions containing 3.4mL IP Dilution Buffer, protein A/G agarose beads, and isotope-matched IgG. Samples were precleared for ?2 hours. Antibody (anti-CTCF, Millipore (07-729, lot 2517762) was prebound to protein A/G agarose beads. Precleared chromatin was added to beads and IPs were performed overnight at 4 degrees C with rotation. Beads were washed once with IP Wash 1 (20mM Tris-HCl pH 8, 2mM EDTA pH 8, 50 mM NaCl, 1% Triton X-100, 0.1% SDS), twice with High Salt Buffer (20 mM Tris-HCl pH 8, 2mM EDTA pH 8, 500 mM NaCl, 1% Triton X-100, 0.01% SDS), once with IP Wash Buffer 2 (10 mM Tris-HCl pH 8, 1mM EDTA pH 8, 0.25 M LiCl, 1% NP-40/Igepal, 1% sodium deoxycholate), and twice with TE (10mM Tris-HCl pH 8, 1mM EDTA pH 8). All washes were performed on ice. Following the final wash, beads were moved to room temperature and eluted twice with 100ul of Elution Buffer (100mM NaHCO3, 1%SDS, prepared fresh) for a final eluate volume of 200ul. The following were added to each sample: 12ul of 5M NaCl, 2ul RNaseA (10mg/ml) and samples were incubated at 65C for ?1 hour. 3ul of Proteinase K (20mg/ml) was added and samples were incubated at 65C overnight. Following overnight incubation, 10ul of 3M sodium acetate pH 5 was added to each sample and DNA was purified using the QIAquick PCR Purification kit per the manufacturer’s instructions. Libraries were prepared according to Illumina's instructions accompanying TruSeq ChIP-seq Sample Preparation Kit. (Part# IP-202-1012). In brief, ChIP-enriched, fragmented DNA was subjected to end repair to generate blunt-end double stranded DNA, adenylation of 3’ ends, and adaptor ligation. Following ligation, SPRIselect (Beckman Coulter) beads were used at 0.9X and 0.6X for left and right side selection, respectively, to obtain an average library target size of ~300 bp. After size selection, fragments were amplified for 16 cycles, and PCR products were purified using Agencourt AMPureXP beads (Beckman Coulter).

Sequencing Platform

instrument_model

NextSeq 500

mm10

Number of total reads

36858795

Reads aligned (%)

97.5

Duplicates removed (%)

18.7

Number of peaks

375 (qval < 1E-05)

mm9

Number of total reads

36858795

Reads aligned (%)

97.2

Duplicates removed (%)

18.7

Number of peaks

446 (qval < 1E-05)