Antigen

Antigen Class

DNase-seq

Antigen

DNase-Seq

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+E2

strain background

129

cell line

G1E-ER4+E2

cell type

inducible GATA-1 rescue subline of G1E cells

cell markers

Ter119+CD71+

treated with

100 nM estradiol for 22h and nocodazole (200 ng/ml) for 7h prior to harvest

Sequenced DNA Library

library_strategy

DNase-Hypersensitivity

library_source

GENOMIC

library_selection

DNase

library_construction_protocol

At harvest, nocodazole-treated and asynchronous cells were cross-linked with 0.1% formaldehyde at room temperature for 10 minutes, then quenched with 1 M glycine. Fixed cells were washed with PBS and resuspended in 1X Cell Lysis Buffer (60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 10 mM Tris pH 7.4, 300 mM sucrose, 0.1 mM EGTA, 0.5 mM PMSF, 0.1% NP40, and 2 μL/mL protease inhibitor cocktail (Sigma)). For mitotic samples, cells were then stained with anti-H3S10Phos antibody (Millipore 04-817) and Dy488 F(ab’)2 anti-rabbit IgG secondary antibody (Jackson 711-485-152), and sorted by FACS for H3S10Phos-positive cells as shown in Fig. 1. DNase I digestion was performed based on protocol from (Cockerill, 1999), with details outlined as follows. 2-10 million asynchronous cells (in Cell Lysis Buffer) or 2 million mitotic cells (collected from the FACS machine in PBS) were resuspended in 50 μL Nuclei Lysis Buffer (300 mM sodium acetate, 5mM EDTA pH 7.4, 0.5% SDS), added to 5 μL of 100 mM CaCl2, equilibrated at room temperature for 10minutes. A range of units of DNase I were added (see DNase-seq library preparation below for the range of units selected for sequencing) and the digestion reaction proceeded for 10 minutes at room temperature, then terminated by adding 350 μL of 0.1 mg/mL proteinase K in Nuclei Lysis Buffer. Samples were gently mixed by inversion, then incubated at 55◦Cfor 5 minutes, then overnight at 65◦Cfor reversal of formaldehyde crosslinks. Additional proteinase K was added to a final concentration of 0.1 mg/mL and incubated at 55◦Cfor 1h. DNA fragments were isolated by phenol-chloroform extraction and ethanol precipitation. DNase-seq library construction was performed as described in (Song and Crawford, 2010), with the following modifications. Standard 0.8% agarose gels were run for 2h at 80V with 5 μL of each sample and stained with ethidium bromide to check extent of chromatin digestion. A range of 3 different DNase I concentrations were chosen for each condition that best matched digestion patterns between conditions. For mitotic samples, this was 2U, 4U, and 8U of DNase. For asynchronous samples, this was between 4U to 40U, adjusted proportionally to the number of cells in the sample. 70 μL of each sample (for each DNase I concentration) were subjected to blunt-end reaction containing 20 μL NEB Buffer 2, 7 μL 10 mM dNTP’s, 6 μL T4 DNA polymerase (NEB M0203), 2 μL BSA (100x), 4 μL 50mMMgCl2, and 95 μL dH2O, incubated at room temperature for 3.25h. 200 μL TE buffer was added and samples placed at 65◦Cfor 15 minutes to deactivate enzyme. Reactions were cleaned up by phenol-chloroform extraction and DNA resuspended in 30 μL 10 mM Tris-Cl. The samples corresponding to the three chosen DNase I concentrations chosen for each condition were measured by nano drop and pooled at equimolar concentrations into a single tube for overnight ligations to the first DNase-seq linker, as per (Song and Crawford, 2010). The one modification from (Song and Crawford, 2010) is that we added a 5’ phosphate to linker 1 to increase ligation efficiency.

Sequencing Platform

instrument_model

Illumina HiSeq 2000

mm10

Number of total reads

74541280

Reads aligned (%)

3.4

Duplicates removed (%)

45.8

Number of peaks

15 (qval < 1E-05)

mm9

Number of total reads

74541280

Reads aligned (%)

3.4

Duplicates removed (%)

46.5

Number of peaks

13 (qval < 1E-05)